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Sample records for exosomes mediates stat3-dependent

  1. Hes1 promotes the IL-22-mediated antimicrobial response by enhancing STAT3-dependent transcription in human intestinal epithelial cells

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    Murano, Tatsuro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Okamoto, Ryuichi, E-mail: rokamoto.gast@tmd.ac.jp [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Ito, Go; Nakata, Toru; Hibiya, Shuji; Shimizu, Hiromichi; Fujii, Satoru; Kano, Yoshihito; Mizutani, Tomohiro; Yui, Shiro; Akiyama-Morio, Junko; Nemoto, Yasuhiro [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Tsuchiya, Kiichiro; Nakamura, Tetsuya [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Department of Advanced GI Therapeutics, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan); Watanabe, Mamoru [Department of Gastroenterology and Hepatology, Graduate School, Tokyo Medical and Dental University, Tokyo (Japan)

    2014-01-17

    Highlights: •Hes1 enhances IL-22-STAT3 signaling in human intestinal epithelial cells. •Hes1 enhances REG family gene induction by IL-22-STAT3 signaling. •Protein level of Hes1 restricts the response to IL-22. •Present regulation of a cytokine signal represents a new mode of Hes1 function. -- Abstract: Notch signaling plays an essential role in the proliferation and differentiation of intestinal epithelial cells (IECs). We have previously shown that Notch signaling is up-regulated in the inflamed mucosa of ulcerative colitis (UC) and thereby plays an indispensable role in tissue regeneration. Here we show that in addition to Notch signaling, STAT3 signaling is highly activated in the inflamed mucosa of UC. Forced expression of the Notch target gene Hes1 dramatically enhanced the IL-22-mediated STAT3-dependent transcription in human IECs. This enhancement of STAT3-dependent transcription was achieved by the extended phosphorylation of STAT3 by Hes1. Microarray analysis revealed that Hes1-mediated enhancement of IL-22-STAT3 signaling significantly increased the induction of genes encoding antimicrobial peptides, such as REG1A, REG3A and REG3G, in human IECs. Conversely, the reduction of Hes1 protein levels with a γ-secretase inhibitor significantly down-regulated the induction of those genes in IECs, resulting in a markedly poor response to IL-22. Our present findings identify a new role for the molecular function of Hes1 in which the protein can interact with cytokine signals and regulate the immune response of IECs.

  2. TIMP-1 mediates the inhibitory effect of interleukin-6 on the proliferation of a hepatocarcinoma cell line in a STAT3-dependent manner

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    S.-Y. Guo

    2007-05-01

    Full Text Available The tissue inhibitor of metalloproteinases (TIMP-1 is a multifunctional protein which is not only an inhibitor of matrix metalloproteinases (MMPs but also to have a possible "cytokine-like" action. Here, we first compared mRNA expression of TIMP-1 and MMP-9 in BEL-7402 (a hepatocellular carcinoma cell line, L-02 (a normal liver cell line and QSG-7701 (a cell line derived from peripheral tissue of liver carcinoma using real-time quantitative RT-PCR. By evaluating the variation of the MMP-9/TIMP-1 ratio as an index of reciprocal changes of the expression of the two genes, we observed that the MMP-9/TIMP-1 ratio was about 13- and 5-fold higher in BEL-7402 than in L-02 and QSG-7701, respectively. Significantly, overexpression of TIMP-1 decreased the MMP-9/TIMP-1 ratio in BEL-7402 and then inhibited the cell growth to 60% and reduced the migration to about 30%. Meanwhile, our data showed that interleukin-6 (IL-6 (100 ng/mL could also inhibited the cell growth of BEL-7402. Further studies indicated that TIMP-1 mediated the inhibitory effect of IL-6 on BEL-7402 cell proliferation in a STAT3-dependent manner, which could further accelerate the expression of the cyclin-dependent kinase inhibitor p21. A dominant negative STAT3 mutant totally abolished IL-6-induced TIMP-1 expression and its biological functions. The present results demonstrate that TIMP-1 may be one of the mediators that regulate the inhibitory effect of IL-6 on BEL-7402 proliferation in which STAT3 signal transduction and p21 up-regulation also play important roles.

  3. Interleukin 17 regulates SHP-2 and IL-17RA/STAT-3 dependent Cyr61, IL-23 and GM-CSF expression and RANKL mediated osteoclastogenesis by fibroblast-like synoviocytes in rheumatoid arthritis.

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    Ganesan, Ramamoorthi; Rasool, Mahaboobkhan

    2017-11-01

    Interleukin (IL)-17 predominately produced by the Th17 cells, plays a crucial role in the fibroblast-like synoviocytes (FLS) mediated disease process of rheumatoid arthritis (RA). IL-17 exerts its pathogenic effects in RA-FLS by IL-17/IL-17RA/STAT-3 signaling. Recent studies have shown that RA-FLS produces SHP-2, Cyr61, IL-23, GM-CSF and RANKL which results in worsening of the disease. However, whether IL-17/IL-17RA/STAT-3 signaling regulates SHP-2, Cyr61, IL-23, GM-CSF and RANKL expressions in RA-FLS remains unknown. In this study, IL-17 treatment dramatically induced the production of Cyr61, IL-23 and GM-CSF in FLS isolated from adjuvant induced arthritis (AA) rats. Conversely, IL-17 mediated production of Cyr61, IL-23 and GM-CSF was abrogated by knockdown of IL-17RA using a small interfering RNA or blockade of STAT-3 activation with S3I-201 in AA-FLS. Interestingly, IL-17 treatment noticeably increased the expression of IL-17RA and SHP-2 in AA-FLS. However, silencing of IL-17RA reversed the effect of IL-17 on the expression of IL-17RA and SHP-2 in AA-FLS. In addition, an increased number of TRAP-positive multinucleated cells were observed in a coculture system consisting of IL-17 treated AA-FLS and rat bone marrow derived monocytes/macrophages. Further, mechanistically we found that IL-17 upregulated RANKL expression in AA-FLS that was dependent on the IL-17/IL-17RA/STAT-3 signaling cascade. Knockdown of IL-17RA or inhibition of STAT-3 activation decreased the IL- 17 induced RANKL expression by AA-FLS and their osteoclastogenic potential. Taken together, our findings demonstrate that IL-17 regulates SHP-2 expression and IL-17RA/STAT-3 dependent production of Cyr61, IL-23, GM-CSF and RANKL in AA-FLS and may reveal a new insight into the pathogenesis of RA. Copyright © 2017 Elsevier Ltd. All rights reserved.

  4. Exosomes as Mediators of the Systemic Adaptations to Endurance Exercise.

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    Safdar, Adeel; Tarnopolsky, Mark A

    2017-05-10

    Habitual endurance exercise training is associated with multisystemic metabolic adaptations that lower the risk of inactivity-associated disorders such as obesity and type 2 diabetes mellitus (T2DM). Identification of complex systemic signaling networks responsible for these benefits are of great interest because of their therapeutic potential in metabolic diseases; however, specific signals that modulate the multisystemic benefits of exercise in multiple tissues and organs are only recently being discovered. Accumulated evidence suggests that muscle and other tissues have an endocrine function and release peptides and nucleic acids into the circulation in response to acute endurance exercise to mediate the multisystemic adaptations. Factors released from skeletal muscle have been termed myokines and we propose that the total of all factors released in response to endurance exercise (including peptides, nucleic acids, and metabolites) be termed, "exerkines." We propose that many of the exerkines are released within extracellular vesicles called exosomes, which regulate peripheral organ cross talk. Exosomes (30-140 nm) and larger microvesicles [MVs] (100-1000 nm) are subcategories of extracellular vesicles that are released into the circulation. Exosomes contain peptides and several nucleic acids (microRNA [miRNA], messenger RNA [mRNA], mitochondrial DNA [mtDNA]) and are involved in intercellular/tissue exchange of their contents. An acute bout of endurance exercise increases circulating exosomes that are hypothesized to mediate organ cross talk to promote systemic adaptation to endurance exercise. Further support for the role of exosomes (and possibly MVs) in mediating the systemic benefits of exercise comes from the fact that the majority of the previously reported myokines/exerkines are found in extracellular vesicles databases (Vesiclepedia and ExoCarta). We propose that exosomes isolated from athletes following exercise or exosomes bioengineered to incorporate

  5. HIF-1-mediated production of exosomes during hypoxia is protective in renal tubular cells.

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    Zhang, Wei; Zhou, Xiangjun; Yao, Qisheng; Liu, Yutao; Zhang, Hao; Dong, Zheng

    2017-10-01

    Exosomes are nano-sized vesicles produced and secreted by cells to mediate intercellular communication. The production and function of exosomes in kidney tissues and cells remain largely unclear. Hypoxia is a common pathophysiological condition in kidneys. This study was designed to characterize exosome production during hypoxia of rat renal proximal tubular cells (RPTCs), investigate the regulation by hypoxia-inducible factor-1 (HIF-1), and determine the effect of the exosomes on ATP-depletion-induced tubular cell injury. Hypoxia did not change the average sizes of exosomes secreted by RPTCs, but it significantly increased exosome production in a time-dependent manner. HIF-1 induction with dimethyloxalylglycine also promoted exosome secretion, whereas pharmacological and genetic suppression of HIF-1 abrogated the increase of exosome secretion under hypoxia. The exosomes from hypoxic RPTCs had inhibitory effects on apoptosis of RPTCs following ATP depletion. The protective effects were lost in the exosomes from HIF-1α knockdown cells. It is concluded that hypoxia stimulates exosome production and secretion in renal tubular cells. The exosomes from hypoxic cells are protective against renal tubular cell injury. HIF-1 mediates exosome production during hypoxia and contributes to the cytoprotective effect of the exosomes. Copyright © 2017 the American Physiological Society.

  6. Identification of novel small molecules that inhibit STAT3-dependent transcription and function.

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    Iryna Kolosenko

    Full Text Available Activation of Signal Transducer and Activator of Transcription 3 (STAT3 has been linked to several processes that are critical for oncogenic transformation, cancer progression, cancer cell proliferation, survival, drug resistance and metastasis. Inhibition of STAT3 signaling has shown a striking ability to inhibit cancer cell growth and therefore, STAT3 has become a promising target for anti-cancer drug development. The aim of this study was to identify novel inhibitors of STAT-dependent gene transcription. A cellular reporter-based system for monitoring STAT3 transcriptional activity was developed which was suitable for high-throughput screening (Z' = 0,8. This system was used to screen a library of 28,000 compounds (the ENAMINE Drug-Like Diversity Set. Following counter-screenings and toxicity studies, we identified four hit compounds that were subjected to detailed biological characterization. Of the four hits, KI16 stood out as the most promising compound, inhibiting STAT3 phosphorylation and transcriptional activity in response to IL6 stimulation. In silico docking studies showed that KI16 had favorable interactions with the STAT3 SH2 domain, however, no inhibitory activity could be observed in the STAT3 fluorescence polarization assay. KI16 inhibited cell viability preferentially in STAT3-dependent cell lines. Taken together, using a targeted, cell-based approach, novel inhibitors of STAT-driven transcriptional activity were discovered which are interesting leads to pursue further for the development of anti-cancer therapeutic agents.

  7. Cardiac fibroblast-derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy

    National Research Council Canada - National Science Library

    Bang, Claudia; Batkai, Sandor; Dangwal, Seema; Gupta, Shashi Kumar; Foinquinos, Ariana; Holzmann, Angelika; Just, Annette; Remke, Janet; Zimmer, Karina; Zeug, Andre; Ponimaskin, Evgeni; Schmiedl, Andreas; Yin, Xiaoke; Mayr, Manuel; Halder, Rashi; Fischer, Andre; Engelhardt, Stefan; Wei, Yuanyuan; Schober, Andreas; Fiedler, Jan; Thum, Thomas

    2014-01-01

    ...; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes...

  8. RNA Exosome Complex-Mediated Control of Redox Status in Pluripotent Stem Cells

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    Maria Skamagki

    2017-10-01

    Full Text Available The RNA exosome complex targets AU-rich element (ARE-containing mRNAs in eukaryotic cells. We identified a transcription factor, ZSCAN10, which binds to the promoters of multiple RNA exosome complex subunits in pluripotent stem cells to maintain subunit gene expression. We discovered that induced pluripotent stem cell clones generated from aged tissue donors (A-iPSC show poor expression of ZSCAN10, leading to poor RNA exosome complex expression, and a subsequent elevation in ARE-containing RNAs, including glutathione peroxidase 2 (Gpx2. Excess GPX2 leads to excess glutathione-mediated reactive oxygen species scavenging activity that blunts the DNA damage response and apoptosis. Expression of ZSCAN10 in A-iPSC recovers RNA exosome gene expression, the DNA damage response, and apoptosis. These findings reveal the central role of ZSCAN10 and the RNA exosome complex in maintaining pluripotent stem cell redox status to support a normal DNA damage response.

  9. Exosomal tetraspanins mediate cancer metastasis by altering host microenvironment.

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    Lu, Jun; Li, Jun; Liu, Shuo; Wang, Teng; Ianni, Alessandro; Bober, Eva; Braun, Thomas; Xiang, Rong; Yue, Shijing

    2017-09-22

    The metastases of malignant tumors develop through a cascade of events. The establishment of a pre-metastatic micro-environment is initiated by communication between tumors and host. Exosomes come into focus as the most potent intercellular communicators playing a pivotal role in this process. Cancer cells release exosomes into the extracellular environment prior to metastasis. Tetraspanin is a type of 4 times transmembrane proteins. It may be involved in cell motility, adhesion, morphogenesis, as well as cell and vesicular membrane fusion. The exosomal tetraspanin network is a molecular scaffold connecting various proteins for signaling transduction. The complex of tetraspanin-integrin determines the recruiting cancer exosomes to pre-metastatic sites. Tetraspanin is a key element for the target cell selection of exosomes uptake that may lead to the reprogramming of target cells. Reprogrammed target cells assist pre-metastatic niche formation. Previous reviews have described the biogenesis, secretion and intercellular interaction of exosomes in various tumors. However, there is a lack of reviews on the topic of exosomal tetraspanin in the context of cancer. In this review, we will describe the main characteristics of exosomal tetraspanin in cancer cells. We will also discuss how the cancer exosomal tetraspanin alters extracellular environment and regulates cancer metastasis.

  10. Exosomes-mediated transfer of long noncoding RNA ZFAS1 promotes gastric cancer progression.

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    Pan, Lei; Liang, Wei; Fu, Min; Huang, Zhen-Hua; Li, Xia; Zhang, Wen; Zhang, Peng; Qian, Hui; Jiang, Peng-Cheng; Xu, Wen-Rong; Zhang, Xu

    2017-06-01

    ZFAS1 is a newly identified long noncoding RNA (lncRNA) that promotes tumor growth and metastasis. Exosomes mediate cellular communications in cancer by transmitting active molecules. The presence of ZFAS1 in the circulating exosomes and the roles of exosomal ZFAS1 in gastric cancer (GC) remains unknown. The aim of this study was to investigate the potential roles of exosomal ZFAS1 in GC. The expression of ZFAS1 was examined in the tumor tissues, serum samples, serum exosomes of GC patients and cell lines using qRT-PCR. The correlation between ZFAS1 expression and the clinicopathological characteristics was analyzed. The characteristics of exosomes were identified using transmission electron microscope (TEM), Nanoparticle Tracking Analysis (NTA), and western blot. The biological roles of ZFAS1 in GC cell growth and mobility were investigated using cell counting, cell colony formation, and transwell migration assay. The potential mechanism of ZFAS1 was demonstrated using flow cytometry, western blot, and qRT-PCR. ZFAS1 expression was elevated in GC cells, tumor tissues, serum and serum exosomes of GC patients. The increased ZFAS1 expression was significantly correlated with lymphatic metastasis and TNM stage. ZFAS1 knockdown inhibited the proliferation and migration of GC cells by suppressing cell cycle progression, inducing apoptosis, and inhibiting epithelial-mesenchymal transition (EMT). On the contrary, ZFAS1 overexpression promoted the proliferation and migration of GC cells. Moreover, ZFAS1 was present in exosomes and could be transmitted by exosomes to enhance GC cell proliferation and migration. ZFAS1 could be delivered by exosomes to promote GC progression, which suggests that ZFAS1 may serve as a potential diagnostic and prognostic biomarker for GC.

  11. Early activation of rat skeletal muscle IL-6/STAT1/STAT3 dependent gene expression in resistance exercise linked to hypertrophy.

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    Gwénaëlle Begue

    Full Text Available Cytokine interleukin-6 (IL-6 is an essential regulator of satellite cell-mediated hypertrophic muscle growth through the transcription factor signal transducer and activator of transcription 3 (STAT3. The importance of this pathway linked to the modulation of myogenic regulatory factors expression in rat skeletal muscle undergoing hypertrophy following resistance exercise, has not been investigated. In this study, the phosphorylation and nuclear localization of STAT3, together with IL-6/STAT3-responsive gene expression, were measured after both a single bout of resistance exercise and 10 weeks of training. Flexor Digitorum Profundus muscle samples from Wistar rats were obtained 2 and 6 hours after a single bout of resistance exercise and 72 h after the last bout of either 2, 4, or 10 weeks of resistance training. We observed an increase in IL-6 and SOCS3 mRNAs concomitant with phosphorylation of STAT1 and STAT3 after 2 and 6 hours of a single bout of exercise (p<0.05. STAT3-dependent early responsive genes such as CyclinD1 and cMyc were also upregulated whereas MyoD and Myf5 mRNAs were downregulated (p<0.05. BrdU-positive satellite cells increased at 2 and 6 hours after exercise (p<0.05. Muscle fiber hypertrophy reached up to 100% after 10 weeks of training and the mRNA expression of Myf5, c-Myc and Cyclin-D1 decreased, whereas IL-6 mRNA remained upregulated. We conclude that the IL-6/STAT1/STAT3 signaling pathway and its responsive genes after a single bout of resistance exercise are an important event regulating the SC pool and behavior involved in muscle hypertrophy after ten weeks of training in rat skeletal muscle.

  12. Upregulation of NLRP3 via STAT3-dependent histone acetylation contributes to painful neuropathy induced by bortezomib.

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    Liu, Cui-Cui; Huang, Zhu-Xi; Li, Xiao; Shen, Kai-Feng; Liu, Meng; Ouyang, Han-Dong; Zhang, Su-Bo; Ruan, Yu-Ting; Zhang, Xiao-Long; Wu, Shao-Ling; Xin, Wen-Jun; Ma, Chao

    2018-01-12

    Painful neuropathy, as a severe side effect of chemotherapeutic bortezomib, is the most common reason for treatment discontinuation. However, the mechanism by which administration of bortezomib leads to painful neuropathy remains unclear. In the present study, we found that application of bortezomib significantly increased the expression of NOD-like receptor family pyrin domain containing 3 (NLRP3) and phosphorylated signal transducer and activator of transcription-3 (STAT3) in dorsal root ganglion (DRG). Intrathecal injection of NLRP3 siRNA significantly prevented the mechanical allodynia induced by bortezomib treatment, and intrathecal injection of recombinant adeno-associated virus vector encoding NLRP3 markedly decreased paw withdrawal threshold of naive rats. Furthermore, the expressions of p-STAT3 were colocalized with NLRP3-positive cells in DRG neurons, and inhibition of STAT3 by intrathecal injection of AAV-Cre-GFP into STAT3 flox/flox mice or inhibitor S3I-201 suppressed the upregulation of NLRP3 and mechanical allodynia induced by bortezomib treatment. Chromatin immunoprecipitation further found that bortezomib increased the recruitment of STAT3, as well as the acetylation of histone H3 and H4, in the NLRP3 promoter region in DRG neurons. Importantly, inhibition of the STAT3 activity by using S3I-201 or DRG local deficiency of STAT3 also significantly prevented the upregulated H3 and H4 acetylation in the NLRP3 promoter region following bortezomib treatment. Altogether, our results suggest that the upregulation of NLRP3 in DRG via STAT3-dependent histone acetylation is critically involved in bortezomib-induced mechanical allodynia. Copyright © 2018. Published by Elsevier Inc.

  13. Exosomes as mediators of platinum resistance in ovarian cancer.

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    Crow, Jennifer; Atay, Safinur; Banskota, Samagya; Artale, Brittany; Schmitt, Sarah; Godwin, Andrew K

    2017-02-14

    Exosomes have been implicated in the cell-cell transfer of oncogenic proteins and genetic material. We speculated this may be one mechanism by which an intrinsically platinum-resistant population of epithelial ovarian cancer (EOC) cells imparts its influence on surrounding tumor cells. To explore this possibility we utilized a platinum-sensitive cell line, A2780 and exosomes derived from its resistant subclones, and an unselected, platinum-resistant EOC line, OVCAR10. A2780 cells demonstrate a ~2-fold increase in viability upon treatment with carboplatin when pre-exposed to exosomes from platinum-resistant cells as compared to controls. This coincided with increased epithelial to mesenchymal transition (EMT). DNA sequencing of EOC cell lines revealed previously unreported somatic mutations in the Mothers Against Decapentaplegic Homolog 4 (SMAD4) within platinum-resistant cells. A2780 cells engineered to exogenously express these SMAD4 mutations demonstrate up-regulation of EMT markers following carboplatin treatment, are more resistant to carboplatin, and release exosomes which impart a ~1.7-fold increase in resistance in naive A2780 recipient cells as compared to controls. These studies provide the first evidence that acquired SMAD4 mutations enhance the chemo-resistance profile of EOC and present a novel mechanism in which exchange of tumor-derived exosomes perpetuates an EMT phenotype, leading to the development of subpopulations of platinum-refractory cells.

  14. Exosomes account for vesicle-mediated transcellular transport of activatable phospholipases and prostaglandins[S

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    Subra, Caroline; Grand, David; Laulagnier, Karine; Stella, Alexandre; Lambeau, Gérard; Paillasse, Michael; De Medina, Philippe; Monsarrat, Bernard; Perret, Bertrand; Silvente-Poirot, Sandrine; Poirot, Marc; Record, Michel

    2010-01-01

    Exosomes are bioactive vesicles released from multivesicular bodies (MVB) by intact cells and participate in intercellular signaling. We investigated the presence of lipid-related proteins and bioactive lipids in RBL-2H3 exosomes. Besides a phospholipid scramblase and a fatty acid binding protein, the exosomes contained the whole set of phospholipases (A2, C, and D) together with interacting proteins such as aldolase A and Hsp 70. They also contained the phospholipase D (PLD) / phosphatidate phosphatase 1 (PAP1) pathway leading to the formation of diglycerides. RBL-2H3 exosomes also carried members of the three phospholipase A2 classes: the calcium-dependent cPLA2-IVA, the calcium-independent iPLA2-VIA, and the secreted sPLA2-IIA and V. Remarkably, almost all members of the Ras GTPase superfamily were present, and incubation of exosomes with GTPγS triggered activation of phospholipase A2 (PLA2)and PLD2. A large panel of free fatty acids, including arachidonic acid (AA) and derivatives such as prostaglandin E2 (PGE2) and 15-deoxy-Δ12,14-prostaglandinJ2 (15-d PGJ2), were detected. We observed that the exosomes were internalized by resting and activated RBL cells and that they accumulated in an endosomal compartment. Endosomal concentrations were in the micromolar range for prostaglandins; i.e., concentrations able to trigger prostaglandin-dependent biological responses. Therefore exosomes are carriers of GTP-activatable phospholipases and lipid mediators from cell to cell. PMID:20424270

  15. Exosome-mediated miR-146a transfer suppresses type I interferon response and facilitates EV71 infection.

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    Yuxuan Fu

    2017-09-01

    Full Text Available Exosomes can transfer genetic materials between cells. Their roles in viral infections are beginning to be appreciated. Researches have shown that exosomes released from virus-infected cells contain a variety of viral and host cellular factors that are able to modulate recipient's cellular response and result in productive infection of the recipient host. Here, we showed that EV71 infection resulted in upregulated exosome secretion and differential packaging of the viral genomic RNA and miR-146a into exosomes. We provided evidence showing that miR-146a was preferentially enriched in exosomes while the viral RNA was not in infected cells. Moreover, the exosomes contained replication-competent EV71 RNA in complex with miR-146a, Ago2, and GW182 and could mediate EV71 transmission independent of virus-specific receptor. The exosomal viral RNA could be transferred to and replicate in a new target cell while the exosomal miR-146a suppressed type I interferon response in the target cell, thus facilitating the viral replication. Additionally, we found that the IFN-stimulated gene factors (ISGs, BST-2/tetherin, were involved in regulating EV71-induced upregulation of exosome secretion. Importantly, in vivo study showed that exosomal viral RNA exhibited differential tissue accumulation as compared to the free virus particles. Together, our findings provide evidence that exosomes secreted by EV71-infected cells selectively packaged high level miR-146a that can be functionally transferred to and facilitate exosomal EV71 RNA to replicate in the recipient cells by suppressing type I interferon response.

  16. Cellular and exosome mediated molecular defense mechanism in bovine granulosa cells exposed to oxidative stress.

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    Saeed-Zidane, Mohammed; Linden, Lea; Salilew-Wondim, Dessie; Held, Eva; Neuhoff, Christiane; Tholen, Ernst; Hoelker, Michael; Schellander, Karl; Tesfaye, Dawit

    2017-01-01

    Various environmental insults including diseases, heat and oxidative stress could lead to abnormal growth, functions and apoptosis in granulosa cells during ovarian follicle growth and oocyte maturation. Despite the fact that cells exposed to oxidative stress are responding transcriptionally, the potential release of transcripts associated with oxidative stress response into extracellular space through exosomes is not yet determined. Therefore, here we aimed to investigate the effect of oxidative stress in bovine granulosa cells in vitro on the cellular and exosome mediated defense mechanisms. Bovine granulosa cells were aspirated from ovarian follicles and cultured in DMEM/F-12 Ham culture medium supplemented with 10% exosome-depleted fetal bovine serum. In the first experiment sub-confluent cells were treated with 5 μM H2O2 for 40 min to induce oxidative stress. Thereafter, cells were subjected to ROS and mitochondrial staining, cell proliferation and cell cycle assays. Furthermore, gene and protein expression analysis were performed in H2O2-challenged versus control group 24 hr post-treatment using qRT-PCR and immune blotting or immunocytochemistry assay, respectively. Moreover, exosomes were isolated from spent media using ultracentrifugation procedure, and subsequently used for RNA isolation and qRT-PCR. In the second experiment, exosomes released by granulosa cells under oxidative stress (StressExo) or those released by granulosa cells without oxidative stress (NormalExo) were co-incubated with bovine granulosa cells in vitro to proof the potential horizontal transfer of defense molecules from exosomes to granulosa cells and investigate any phenotype changes. Exposure of bovine granulosa cells to H2O2 induced the accumulation of ROS, reduced mitochondrial activity, increased expression of Nrf2 and its downstream antioxidant genes (both mRNA and protein), altered the cell cycle transitions and induced cellular apoptosis. Granulosa cells exposed to oxidative

  17. Exosome-mediated microRNA transfer plays a role in radiation-induced bystander effect.

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    Xu, Shuai; Wang, Jufang; Ding, Nan; Hu, Wentao; Zhang, Xurui; Wang, Bing; Hua, Junrui; Wei, Wenjun; Zhu, Qiyun

    2015-01-01

    Bystander effects can be induced through cellular communication between irradiated cells and non-irradiated cells. The signals that mediate this cellular communication, such as cytokines, reactive oxygen species, nitric oxide and even microRNAs, can be transferred between cells via gap junctions or extracellular medium. We have previously reported that miR-21, a well described DDR (DNA damage response) microRNA, is involved in radiation-induced bystander effects through a medium-mediated way. However, the mechanisms of the microRNA transfer have not been elucidated in details. In the present study, it was found that exosomes isolated from irradiated conditioned medium could induce bystander effects. Furthermore, we demonstrated plenty of evidences that miR-21, which is up-regulated as a result of mimic transfection or irradiation, can be transferred from donor or irradiated cells into extracellular medium and subsequently get access to the recipient or bystander cells through exosomes to induce bystander effects. Inhibiting the miR-21 expression in advance can offset the bystander effects to some extent. From all of these results, it can be concluded that the exosome-mediated microRNA transfer plays an important role in the radiation-induced bystander effects. These findings provide new insights into the functions of microRNAs and the cellular communication between the directly irradiated cells and the non-irradiated cells.

  18. Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect.

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    Michelle Le

    Full Text Available The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal.The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells were not directly irradiated but were exposed to the emitted UV biophotons. Medium was subsequently harvested from UV-exposed bystander cells. The exosomes extracted from this medium were incubated with reporter cell populations. These reporter cells were then assayed for clonogenic survival and mitochondrial membrane potential with and without prior treatment of the exosomes with RNase.Clonogenic cell survival was significantly reduced in reporter cells incubated with exosomes extracted from cells exposed to secondarily-emitted UV. These exosomes also induced significant mitochondrial membrane depolarization in receiving reporter cells. Conversely, exosomes extracted from non-UV-exposed cells did not produce bystander effects in reporter cells. The treatment of exosomes with RNase prior to their incubation with reporter cells effectively abolished bystander effects in reporter cells and this suggests a role for RNA in mediating the bystander response elicited by UV biophotons and their produced exosomes.This study supports a role for exosomes released from UV biophoton-exposed bystander cells in eliciting bystander responses and also indicates a reconciliation between the UV-mediated bystander effect and the bystander effect which has been suggested in the literature to be mediated by soluble factors.

  19. Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect.

    Science.gov (United States)

    Le, Michelle; Fernandez-Palomo, Cristian; McNeill, Fiona E; Seymour, Colin B; Rainbow, Andrew J; Mothersill, Carmel E

    2017-01-01

    The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV) biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal. The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells were not directly irradiated but were exposed to the emitted UV biophotons. Medium was subsequently harvested from UV-exposed bystander cells. The exosomes extracted from this medium were incubated with reporter cell populations. These reporter cells were then assayed for clonogenic survival and mitochondrial membrane potential with and without prior treatment of the exosomes with RNase. Clonogenic cell survival was significantly reduced in reporter cells incubated with exosomes extracted from cells exposed to secondarily-emitted UV. These exosomes also induced significant mitochondrial membrane depolarization in receiving reporter cells. Conversely, exosomes extracted from non-UV-exposed cells did not produce bystander effects in reporter cells. The treatment of exosomes with RNase prior to their incubation with reporter cells effectively abolished bystander effects in reporter cells and this suggests a role for RNA in mediating the bystander response elicited by UV biophotons and their produced exosomes. This study supports a role for exosomes released from UV biophoton-exposed bystander cells in eliciting bystander responses and also indicates a reconciliation between the UV-mediated bystander effect and the bystander effect which has been suggested in the literature to be mediated by soluble factors.

  20. Cardiac fibroblast–derived microRNA passenger strand-enriched exosomes mediate cardiomyocyte hypertrophy

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    Bang, Claudia; Batkai, Sandor; Dangwal, Seema; Gupta, Shashi Kumar; Foinquinos, Ariana; Holzmann, Angelika; Just, Annette; Remke, Janet; Zimmer, Karina; Zeug, Andre; Ponimaskin, Evgeni; Schmiedl, Andreas; Yin, Xiaoke; Mayr, Manuel; Halder, Rashi; Fischer, Andre; Engelhardt, Stefan; Wei, Yuanyuan; Schober, Andreas; Fiedler, Jan; Thum, Thomas

    2014-01-01

    In response to stress, the heart undergoes extensive cardiac remodeling that results in cardiac fibrosis and pathological growth of cardiomyocytes (hypertrophy), which contribute to heart failure. Alterations in microRNA (miRNA) levels are associated with dysfunctional gene expression profiles associated with many cardiovascular disease conditions; however, miRNAs have emerged recently as paracrine signaling mediators. Thus, we investigated a potential paracrine miRNA crosstalk between cardiac fibroblasts and cardiomyocytes and found that cardiac fibroblasts secrete miRNA-enriched exosomes. Surprisingly, evaluation of the miRNA content of cardiac fibroblast–derived exosomes revealed a relatively high abundance of many miRNA passenger strands (“star” miRNAs), which normally undergo intracellular degradation. Using confocal imaging and coculture assays, we identified fibroblast exosomal–derived miR-21_3p (miR-21*) as a potent paracrine-acting RNA molecule that induces cardiomyocyte hypertrophy. Proteome profiling identified sorbin and SH3 domain-containing protein 2 (SORBS2) and PDZ and LIM domain 5 (PDLIM5) as miR-21* targets, and silencing SORBS2 or PDLIM5 in cardiomyocytes induced hypertrophy. Pharmacological inhibition of miR-21* in a mouse model of Ang II–induced cardiac hypertrophy attenuated pathology. These findings demonstrate that cardiac fibroblasts secrete star miRNA–enriched exosomes and identify fibroblast-derived miR-21* as a paracrine signaling mediator of cardiomyocyte hypertrophy that has potential as a therapeutic target. PMID:24743145

  1. Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells

    Science.gov (United States)

    Domenis, Rossana; Cesselli, Daniela; Toffoletto, Barbara; Bourkoula, Evgenia; Caponnetto, Federica; Manini, Ivana; Beltrami, Antonio Paolo; Ius, Tamara; Skrap, Miran; Di Loreto, Carla

    2017-01-01

    A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression. PMID:28107450

  2. Systemic T Cells Immunosuppression of Glioma Stem Cell-Derived Exosomes Is Mediated by Monocytic Myeloid-Derived Suppressor Cells.

    Science.gov (United States)

    Domenis, Rossana; Cesselli, Daniela; Toffoletto, Barbara; Bourkoula, Evgenia; Caponnetto, Federica; Manini, Ivana; Beltrami, Antonio Paolo; Ius, Tamara; Skrap, Miran; Di Loreto, Carla; Gri, Giorgia

    2017-01-01

    A major contributing factor to glioma development and progression is its ability to evade the immune system. Nano-meter sized vesicles, exosomes, secreted by glioma-stem cells (GSC) can act as mediators of intercellular communication to promote tumor immune escape. Here, we investigated the immunomodulatory properties of GCS-derived exosomes on different peripheral immune cell populations. Healthy donor peripheral blood mononuclear cells (PBMCs) stimulated with anti-CD3, anti-CD28 and IL-2, were treated with GSC-derived exosomes. Phenotypic characterization, cell proliferation, Th1/Th2 cytokine secretion and intracellular cytokine production were analysed by distinguishing among effector T cells, regulatory T cells and monocytes. In unfractionated PBMCs, GSC-derived exosomes inhibited T cell activation (CD25 and CD69 expression), proliferation and Th1 cytokine production, and did not affect cell viability or regulatory T-cell suppression ability. Furthermore, exosomes were able to enhance proliferation of purified CD4+ T cells. In PBMCs culture, glioma-derived exosomes directly promoted IL-10 and arginase-1 production and downregulation of HLA-DR by unstimulated CD14+ monocytic cells, that displayed an immunophenotype resembling that of monocytic myeloid-derived suppressor cells (Mo-MDSCs). Importantly, the removal of CD14+ monocytic cell fraction from PBMCs restored T-cell proliferation. The same results were observed with exosomes purified from plasma of glioblastoma patients. Our results indicate that glioma-derived exosomes suppress T-cell immune response by acting on monocyte maturation rather than on direct interaction with T cells. Selective targeting of Mo-MDSC to treat glioma should be considered with regard to how immune cells allow the acquirement of effector functions and therefore counteracting tumor progression.

  3. Cardiomyocytes mediate anti-angiogenesis in type 2 diabetic rats through the exosomal transfer of miR-320 into endothelial cells

    Science.gov (United States)

    Wang, Xiaohong; Huang, Wei; Liu, Guansheng; Cai, Wenfeng; Millard, Ronald W.; Wang, Yigang; Chang, Jiang; Peng, Tianqing; Fan, Guo-Chang

    2014-01-01

    Exosomes, nano-vesicles naturally released from living cells, have been well recognized to play critical roles in mediating cell-to-cell communication. Given that diabetic hearts exhibit insufficient angiogenesis, it is significant to test whether diabetic cardiomyocyte-derived exosomes possess any capacity in regulating angiogenesis. In this study, we first observed that both proliferation and migration of mouse cardiac endothelial cells (MCECs) were inhibited when co-cultured with cardiomyocytes isolated from adult Goto-Kakizaki (GK) rats, a commonly used animal model of type 2 diabetes. However, GK-myocyte-mediated anti-angiogenic effects were negated upon addition of GW4869, an inhibitor of exosome formation/release, into the co-cultures. Next, exosomes were purified from the myocyte culture supernatants by differential centrifugation. While exosomes derived from GK myocytes (GK-exosomes) displayed similar size and molecular markers (CD63 and CD81) to those originated from the control Wistar rat myocytes (WT-exosomes), their regulatory role in angiogenesis is opposite. We observed that the MCEC proliferation, migration and tube-like formation were inhibited by GK-exosomes, but were promoted by WT-exosomes. Mechanistically, we found that GK-exosomes encapsulated higher levels of miR-320 and lower levels of miR-126 compared to WT-exosomes. Furthermore, GK-exosomes were effectively taken up by MCECs and delivered miR-320. In addition, transportation of miR-320 from myocytes to MCECs could be blocked by GW4869. Importantly, the exosomal miR-320 functionally down-regulated its target genes (IGF-1, Hsp20 and Ets2) in recipient MCECs, and overexpression of miR-320 inhibited MCEC migration and tube formation. GK exosome-mediated inhibitory effects on angiogenesis were removed by knockdown of miR-320. Together, these data indicate that cardiomyocytes exert an anti-angiogenic function in type 2 diabetic rats through exosomal transfer of miR-320 into endothelial cells

  4. Release of luminal exosomes contributes to TLR4-mediated epithelial antimicrobial defense.

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    Guoku Hu

    Full Text Available Exosomes are membranous nanovesicles released by most cell types from multi-vesicular endosomes. They are speculated to transfer molecules to neighboring or distant cells and modulate many physiological and pathological procedures. Exosomes released from the gastrointestinal epithelium to the basolateral side have been implicated in antigen presentation. Here, we report that luminal release of exosomes from the biliary and intestinal epithelium is increased following infection by the protozoan parasite Cryptosporidium parvum. Release of exosomes involves activation of TLR4/IKK2 signaling through promoting the SNAP23-associated vesicular exocytotic process. Downregulation of let-7 family miRNAs by activation of TLR4 signaling increases SNAP23 expression, coordinating exosome release in response to C. parvum infection. Intriguingly, exosomes carry antimicrobial peptides of epithelial cell origin, including cathelicidin-37 and beta-defensin 2. Activation of TLR4 signaling enhances exosomal shuttle of epithelial antimicrobial peptides. Exposure of C. parvum sporozoites to released exosomes decreases their viability and infectivity both in vitro and ex vivo. Direct binding to the C. parvum sporozoite surface is required for the anti-C. parvum activity of released exosomes. Biliary epithelial cells also increase exosomal release and display exosome-associated anti-C. parvum activity following LPS stimulation. Our data indicate that TLR4 signaling regulates luminal exosome release and shuttling of antimicrobial peptides from the gastrointestinal epithelium, revealing a new arm of mucosal immunity relevant to antimicrobial defense.

  5. Exosomes derived from mature dendritic cells increase endothelial inflammation and atherosclerosis via membrane TNF-α mediated NF-κB pathway.

    Science.gov (United States)

    Gao, Wei; Liu, Haibo; Yuan, Jie; Wu, Chaoneng; Huang, Dong; Ma, Yuanji; Zhu, Jianbing; Ma, Leilei; Guo, Junjie; Shi, Hongtao; Zou, Yunzeng; Ge, Junbo

    2016-12-01

    Whether dendritic cell (DC) derived exosomes play a role in the progression of endothelial inflammation and atherosclerosis remains unclear. Using a transwell system and exosome release inhibitor GW4869, we demonstrated that mature DCs contributed to endothelial inflammation and exosomes were involved in the process. To further confirm this finding, we isolated exosomes from bone marrow dendritic cell (BMDC) culture medium (named DC-exos) and stimulated human umbilical vein endothelial cell (HUVEC) with these DC-exos. We observed that mature DC-exos increased HUVEC inflammation through NF-κB pathway in a manner similar to that of lipopolysaccharide. After a protein array analysis of exosomes, we identified and confirmed tumour necrosis factor (TNF)-α on exosome membrane being the trigger of NF-κB pathway in HUVECs. We then performed an in vivo study and found that the aorta endothelial of mice could uptake intravenously injected exosomes and was activated by these exosomes. After a period of 12 weeks of mature DC-exos injection into ApoE-/- mice, the atherosclerotic lesions significantly increased. Our study demonstrates that mature DCs derived exosomes increase endothelial inflammation and atherosclerosis via membrane TNF-α mediated NF-κB pathway. This finding extends our knowledge on how DCs affect inflammation and provides a potential method to prevent endothelial inflammation and atherosclerosis. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  6. Glial Scar Borders Are Formed by Newly Proliferated, Elongated Astrocytes That Interact to Corral Inflammatory and Fibrotic Cells via STAT3-Dependent Mechanisms after Spinal Cord Injury

    Science.gov (United States)

    Anderson, Mark A.; Song, Bingbing; Levine, Jaclynn; Fernandez, Ana; Gray-Thompson, Zachary; Ao, Yan

    2013-01-01

    Astroglial scars surround damaged tissue after trauma, stroke, infection, or autoimmune inflammation in the CNS. They are essential for wound repair, but also interfere with axonal regrowth. A better understanding of the cellular mechanisms, regulation, and functions of astroglial scar formation is fundamental to developing safe interventions for many CNS disorders. We used wild-type and transgenic mice to quantify and dissect these parameters. Adjacent to crush spinal cord injury (SCI), reactive astrocytes exhibited heterogeneous phenotypes as regards proliferation, morphology, and chemistry, which all varied with distance from lesions. Mature scar borders at 14 d after SCI consisted primarily of newly proliferated astroglia with elongated cell processes that surrounded large and small clusters of inflammatory, fibrotic, and other cells. During scar formation from 5 to 14 d after SCI, cell processes deriving from different astroglia associated into overlapping bundles that quantifiably reoriented and organized into dense mesh-like arrangements. Selective deletion of STAT3 from astroglia quantifiably disrupted the organization of elongated astroglia into scar borders, and caused a failure of astroglia to surround inflammatory cells, resulting in increased spread of these cells and neuronal loss. In cocultures, wild-type astroglia spontaneously corralled inflammatory or fibromeningeal cells into segregated clusters, whereas STAT3-deficient astroglia failed to do so. These findings demonstrate heterogeneity of reactive astroglia and show that scar borders are formed by newly proliferated, elongated astroglia, which organize via STAT3-dependent mechanisms to corral inflammatory and fibrotic cells into discrete areas separated from adjacent tissue that contains viable neurons. PMID:23904622

  7. Trichomonas vaginalis Exosomes Deliver Cargo to Host Cells and Mediate Host∶Parasite Interactions

    Science.gov (United States)

    Twu, Olivia; Lustig, Gila; Stevens, Grant C.; Vashisht, Ajay A.; Wohlschlegel, James A.

    2013-01-01

    Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization. PMID:23853596

  8. Trichomonas vaginalis exosomes deliver cargo to host cells and mediate host∶parasite interactions.

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    Olivia Twu

    Full Text Available Trichomonas vaginalis is a common sexually transmitted parasite that colonizes the human urogential tract where it remains extracellular and adheres to epithelial cells. Infections range from asymptomatic to highly inflammatory, depending on the host and the parasite strain. Here, we use a combination of methodologies including cell fractionation, immunofluorescence and electron microscopy, RNA, proteomic and cytokine analyses and cell adherence assays to examine pathogenic properties of T. vaginalis. We have found that T.vaginalis produces and secretes microvesicles with physical and biochemical properties similar to mammalian exosomes. The parasite-derived exosomes are characterized by the presence of RNA and core, conserved exosomal proteins as well as parasite-specific proteins. We demonstrate that T. vaginalis exosomes fuse with and deliver their contents to host cells and modulate host cell immune responses. Moreover, exosomes from highly adherent parasite strains increase the adherence of poorly adherent parasites to vaginal and prostate epithelial cells. In contrast, exosomes from poorly adherent strains had no measurable effect on parasite adherence. Exosomes from parasite strains that preferentially bind prostate cells increased binding of parasites to these cells relative to vaginal cells. In addition to establishing that parasite exosomes act to modulate host∶parasite interactions, these studies are the first to reveal a potential role for exosomes in promoting parasite∶parasite communication and host cell colonization.

  9. Activated human T cells secrete exosomes that participate in IL-2 mediated immune response signaling.

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    Jessica Wahlgren

    Full Text Available It has previously been shown that nano-meter sized vesicles (30-100 nm, exosomes, secreted by antigen presenting cells can induce T cell responses thus showing the potential of exosomes to be used as immunological tools. Additionally, activated CD3⁺ T cells can secrete exosomes that have the ability to modulate different immunological responses. Here, we investigated what effects exosomes originating from activated CD3⁺ T cells have on resting CD3⁺ T cells by studying T cell proliferation, cytokine production and by performing T cell and exosome phenotype characterization. Human exosomes were generated in vitro following CD3⁺ T cell stimulation with anti-CD28, anti-CD3 and IL-2. Our results show that exosomes purified from stimulated CD3⁺ T cells together with IL-2 were able to generate proliferation in autologous resting CD3⁺ T cells. The CD3⁺ T cells stimulated with exosomes together with IL-2 had a higher proportion of CD8⁺ T cells and had a different cytokine profile compared to controls. These results indicate that activated CD3⁺ T cells communicate with resting autologous T cells via exosomes.

  10. Programmed Fetal Membrane Senescence and Exosome-Mediated Signaling: A Mechanism Associated With Timing of Human Parturition

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    Ramkumar Menon

    2017-08-01

    Full Text Available Human parturition is an inflammatory process that involves both fetal and maternal compartments. The precise immune cell interactions have not been well delineated in human uterine tissues during parturition, but insights into human labor initiation have been informed by studies in animal models. Unfortunately, the timing of parturition relative to fetal maturation varies among viviparous species—indicative of different phylogenetic clocks and alarms—but what is clear is that important common pathways must converge to control the birth process. Herein, we hypothesize a novel signaling mechanism initiated by human fetal membrane aging and senescence-associated inflammation. Programmed events of fetal membrane aging coincide with fetal growth and organ maturation. Mechanistically, senescence involves in telomere shortening and activation of p38 mitogen-activated signaling kinase resulting in aging-associated phenotypic transition. Senescent tissues release inflammatory signals that are propagated via exosomes to cause functional changes in maternal uterine tissues. In vitro, oxidative stress causes increased release of inflammatory mediators (senescence-associated secretory phenotype and damage-associated molecular pattern markers that can be packaged inside the exosomes. These exosomes traverse through tissues layers, reach maternal tissues to increase overall inflammatory load transitioning them from a quiescent to active state. Animal model studies have shown that fetal exosomes can travel from fetal to the maternal side. Thus, aging fetal membranes and membrane-derived exosomes cargo fetal signals to the uterus and cervix and may trigger parturition. This review highlights a novel hypothesis in human parturition research based on data from ongoing research using human fetal membrane model system.

  11. Immune-induced fever is mediated by IL-6 receptors on brain endothelial cells coupled to STAT3-dependent induction of brain endothelial prostaglandin synthesis.

    Science.gov (United States)

    Eskilsson, Anna; Mirrasekhian, Elahe; Dufour, Sylvie; Schwaninger, Markus; Engblom, David; Blomqvist, Anders

    2014-11-26

    The cytokine IL-6, which is released upon peripheral immune challenge, is critical for the febrile response, but the mechanism by which IL-6 is pyrogenic has remained obscure. Here we generated mice with deletion of the membrane bound IL-6 receptor α (IL-6Rα) on neural cells, on peripheral nerves, on fine sensory afferent fibers, and on brain endothelial cells, respectively, and examined its role for the febrile response to peripherally injected lipopolysaccharide. We show that IL-6Rα on neural cells, peripheral nerves, and fine sensory afferents are dispensable for the lipopolysaccharide-induced fever, whereas IL-6Rα in the brain endothelium plays an important role. Hence deletion of IL-6Rα on brain endothelial cells strongly attenuated the febrile response, and also led to reduced induction of the prostaglandin synthesizing enzyme Cox-2 in the hypothalamus, the temperature-regulating center in the brain, as well as reduced expression of SOCS3, suggesting involvement of the STAT signaling pathway. Furthermore, deletion of STAT3 in the brain endothelium also resulted in attenuated fever. These data show that IL-6, when endogenously released during systemic inflammation, is pyrogenic by binding to IL-6Rα on brain endothelial cells to induce prostaglandin synthesis in these cells, probably in concerted action with other peripherally released cytokines. Copyright © 2014 the authors 0270-6474/14/3415957-05$15.00/0.

  12. Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction

    Science.gov (United States)

    Yang, Yinli; Han, Qiuju; Hou, Zhaohua; Zhang, Cai; Tian, Zhigang; Zhang, Jian

    2017-01-01

    Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus (HBV) infection remain unclear. Here, we report that exosomes present in the sera of chronic hepatitis B (CHB) patients contained both HBV nucleic acids and HBV proteins, and transferred HBV to hepatocytes in an active manner. Notably, HBV nucleic acids were detected in natural killer (NK) cells from both CHB patients and healthy donors after exposure to HBV-positive exosomes. Through real-time fluorescence microscopy and flow cytometry, 1,1'-dioctadecyl-3,3,3',3',-tetramethylindodicarbocyanine, 4-chlorobenzenesulfnate salt (DiD)-labeled exosomes were observed to interact with NK cells and to be taken up by NK cells, which was enhanced by transforming growth factor-β treatment. Furthermore, HBV-positive exosomes impaired NK-cell functions, including interferon (IFN)-γ production, cytolytic activity, NK-cell proliferation and survival, as well as the responsiveness of the cells to poly (I:C) stimulation. HBV infection suppressed the expression of pattern-recognition receptors, especially retinoic acid inducible gene I (RIG-I), on NK cells, resulting in the dampening of the nuclear factor κB(NF-κB) and p38 mitogen-activated protein kinase pathways. Our results highlight a previously unappreciated role of exosomes in HBV transmission and NK-cell dysfunction during CHB infection. PMID:27238466

  13. Exosomes mediate hepatitis B virus (HBV) transmission and NK-cell dysfunction.

    Science.gov (United States)

    Yang, Yinli; Han, Qiuju; Hou, Zhaohua; Zhang, Cai; Tian, Zhigang; Zhang, Jian

    2017-05-01

    Evidence suggests that exosomes can transfer genetic material between cells. However, their roles in hepatitis B virus (HBV) infection remain unclear. Here, we report that exosomes present in the sera of chronic hepatitis B (CHB) patients contained both HBV nucleic acids and HBV proteins, and transferred HBV to hepatocytes in an active manner. Notably, HBV nucleic acids were detected in natural killer (NK) cells from both CHB patients and healthy donors after exposure to HBV-positive exosomes. Through real-time fluorescence microscopy and flow cytometry, 1,1'-dioctadecyl-3,3,3',3',-tetramethylindodicarbocyanine, 4-chlorobenzenesulfnate salt (DiD)-labeled exosomes were observed to interact with NK cells and to be taken up by NK cells, which was enhanced by transforming growth factor-β treatment. Furthermore, HBV-positive exosomes impaired NK-cell functions, including interferon (IFN)-γ production, cytolytic activity, NK-cell proliferation and survival, as well as the responsiveness of the cells to poly (I:C) stimulation. HBV infection suppressed the expression of pattern-recognition receptors, especially retinoic acid inducible gene I (RIG-I), on NK cells, resulting in the dampening of the nuclear factor κB(NF-κB) and p38 mitogen-activated protein kinase pathways. Our results highlight a previously unappreciated role of exosomes in HBV transmission and NK-cell dysfunction during CHB infection.

  14. Exosomes mediate interepithelial transfer of functional P-glycoprotein in chronic rhinosinusitis with nasal polyps.

    Science.gov (United States)

    Nocera, Angela L; Miyake, Marcel M; Seifert, Philip; Han, Xue; Bleier, Benjamin S

    2017-09-01

    P-glycoprotein (P-gp) drives type-2 helper T-cell inflammation in chronic rhinosinusitis with nasal polyps (CRSwNP) through unknown posttranslational mechanisms of overexpression. A recent randomized clinical trial demonstrated that inhibition of P-gp was as effective as oral steroids and biologics in treating CRSwNP. Exosomes are 30- to 150-nm vesicles capable of intercellular membrane protein transfer. The aims of this study were 1) to determine whether CRSwNP mucus exosomes are enriched with P-gp, and 2) whether exosomal P-gp can be functionally transferred to autologous epithelial cells as a putative mechanism for the proinflammatory overexpression of P-gp in CRSwNP. Institutional review board-approved study in CRSwNP and control patients (n = 10 per group). P-gp content of purified mucus exosomes was characterized by transmission electron microscopy and enzyme-linked immunosorbent assay. Epithelial transfer of exosomal P-gp was determined by time-lapse fluorescent microscopy and calcein acetoxymethylester functional P-gp assay. CD63+/P-gp+ exosomes were detected in both groups. P-gp was significantly enriched in CRSwNP exosomes relative to control (median 198.5; interquartile range 123.6-270.5 vs. 74.4; 41.3-95.0 pcg P-gp/10(9) exosomes, P = 0.002). Exosomes were absorbed by epithelial cells within 10 minutes, resulting in a significant increase in P-gp activity in CRSwNP patients relative to control (P = 0.006). Here we demonstrate the presence and P-gp enrichment of mucus-derived exosomes, or rhinosomes, in CRSwNP. These rhinosomes are capable of rapid intercellular transfer of P-gp, leading to increased P-gp function within recipient cells. This represents a novel mechanism for maintaining P-gp overexpression in CRSwNP, and more generally for interepithelial transfer of other proteins between mucosal epithelial cells. NA. Laryngoscope, 127:E295-E300, 2017. © 2017 The American Laryngological, Rhinological and Otological Society, Inc.

  15. UCH-L1-containing exosomes mediate chemotherapeutic resistance transfer in breast cancer.

    Science.gov (United States)

    Ning, Kuan; Wang, Teng; Sun, Xu; Zhang, Pengfei; Chen, Yun; Jin, Jian; Hua, Dong

    2017-06-01

    Chemotherapy resistance has become a serious challenge in the treatment of breast cancer. Previous studies showed cells can transfer proteins, including those responsible for drug resistance to adjacent cells via exosomes. The switches of drug resistance via exosomes transfer were assessed by CellTiter-Blue Viability assay, flow cytometry, and immunostaining analysis. Relative protein levels of Ubiquitin carboxyl terminal hydrolase-L1 (UCH-L1), P-glycoprotein (P-gp), extracellular-signal regulated protein kinase1/2 (ERK1/2), and phospho-extracellular-signal regulated protein kinase1/2 (p-ERK1/2) were measured by Western blot. Immunohistochemistry was performed on 93 breast cancer samples to assess the associations of UCH-L1 levels with immunofluorescence value of UCH-L1 in circulating exosomes. The Adriamycin-resistant human breast cancer cells (MCF7/ADM) secreted exosomes carrying UCH-L1 and P-gp proteins into the extracellular microenvironment then integrated into Adriamycin-sensitive human breast cancer cells (MCF7/WT) in a time-dependent manner, transferring the chemoresistance phenotype. Notably, in blood samples from patients with breast cancer, the level of exosomes carrying UCH-L1 before chemotherapy was significantly negatively correlated with prognosis. Our study demonstrated that UCH-L1-containing exosomes can transfer chemoresistance to recipient cells and these exosomes may be useful as non-invasive diagnostic biomarkers for detection of chemoresitance in breast cancer patients, achieving more effective and individualized chemotherapy. © 2017 Wiley Periodicals, Inc.

  16. Post-translational modifications of exosomal proteins.

    Science.gov (United States)

    Moreno-Gonzalo, Olga; Villarroya-Beltri, Carolina; Sánchez-Madrid, Francisco

    2014-01-01

    Exosomes mediate intercellular communication and participate in many cell processes such as cancer progression, immune activation or evasion, and the spread of infection. Exosomes are small vesicles secreted to the extracellular environment through the release of intraluminal vesicles contained in multivesicular bodies (MVBs) upon the fusion of these MVBs with the plasma membrane. The composition of exosomes is not random, suggesting that the incorporation of cargo into them is a regulated process. However, the mechanisms that control the sorting of protein cargo into exosomes are currently elusive. Here, we review the post-translational modifications detected in exosomal proteins, and discuss their possible role in their specific sorting into exosomes.

  17. Characterization of human thymic exosomes

    OpenAIRE

    Gabriel Skogberg; Judith Gudmundsdottir; Sjoerd van der Post; Kerstin Sandström; Sören Bruhn; Mikael Benson; Lucia Mincheva-Nilsson; Vladimir Baranov; Esbjörn Telemo; Olov Ekwall

    2013-01-01

    Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytom...

  18. Exosomal signaling during hypoxia mediates microvascular endothelial cell migration and vasculogenesis.

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    Carlos Salomon

    Full Text Available Vasculogenesis and angiogenesis are critical processes in fetal circulation and placental vasculature development. Placental mesenchymal stem cells (pMSC are known to release paracrine factors (some of which are contained within exosomes that promote angiogenesis and cell migration. The aims of this study were: to determine the effects of oxygen tension on the release of exosomes from pMSC; and to establish the effects of pMSC-derived exosomes on the migration and angiogenic tube formation of placental microvascular endothelial cells (hPMEC. pMSC were isolated from placental villi (8-12 weeks of gestation, n = 6 and cultured under an atmosphere of 1%, 3% or 8% O2. Cell-conditioned media were collected and exosomes (exo-pMSC isolated by differential and buoyant density centrifugation. The dose effect (5-20 µg exosomal protein/ml of pMSC-derived exosomes on hPMEC migration and tube formation were established using a real-time, live-cell imaging system (Incucyte™. The exosome pellet was resuspended in PBS and protein content was established by mass spectrometry (MS. Protein function and canonical pathways were identified using the PANTHER program and Ingenuity Pathway Analysis, respectively. Exo-pMSC were identified, by electron microscopy, as spherical vesicles, with a typical cup-shape and diameters around of 100 nm and positive for exosome markers: CD63, CD9 and CD81. Under hypoxic conditions (1% and 3% O2 exo-pMSC released increased by 3.3 and 6.7 folds, respectively, when compared to the controls (8% O2; p<0.01. Exo-pMSC increased hPMEC migration by 1.6 fold compared to the control (p<0.05 and increased hPMEC tube formation by 7.2 fold (p<0.05. MS analysis identified 390 different proteins involved in cytoskeleton organization, development, immunomodulatory, and cell-to-cell communication. The data obtained support the hypothesis that pMSC-derived exosomes may contribute to placental vascular adaptation to low oxygen tension under both

  19. Exosomes Mediate Mobilization of Autocrine Wnt10b to Promote Axonal Regeneration in the Injured CNS

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    Nardos G. Tassew

    2017-07-01

    Full Text Available Developing strategies that promote axonal regeneration within the injured CNS is a major therapeutic challenge, as axonal outgrowth is potently inhibited by myelin and the glial scar. Although regeneration can be achieved using the genetic deletion of PTEN, a negative regulator of the mTOR pathway, this requires inactivation prior to nerve injury, thus precluding therapeutic application. Here, we show that, remarkably, fibroblast-derived exosomes (FD exosomes enable neurite growth on CNS inhibitory proteins. Moreover, we demonstrate that, upon treatment with FD exosomes, Wnt10b is recruited toward lipid rafts and activates mTOR via GSK3β and TSC2. Application of FD exosomes shortly after optic nerve injury promoted robust axonal regeneration, which was strongly reduced in Wnt10b-deleted animals. This work uncovers an intercellular signaling pathway whereby FD exosomes mobilize an autocrine Wnt10b-mTOR pathway, thereby awakening the intrinsic capacity of neurons for regeneration, an important step toward healing the injured CNS.

  20. MIRNAS in Astrocyte-Derived Exosomes as Possible Mediators of Neuronal Plasticity

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    Carlos Lafourcade

    2016-01-01

    Full Text Available Astrocytes use gliotransmitters to modulate neuronal function and plasticity. However, the role of small extracellular vesicles, called exosomes, in astrocyte-to-neuron signaling is mostly unknown. Exosomes originate in multivesicular bodies of parent cells and are secreted by fusion of the multivesicular body limiting membrane with the plasma membrane. Their molecular cargo, consisting of RNA species, proteins, and lipids, is in part cell type and cell state specific. Among the RNA species transported by exosomes, microRNAs (miRNAs are able to modify gene expression in recipient cells. Several miRNAs present in astrocytes are regulated under pathological conditions, and this may have far-reaching consequences if they are loaded in exosomes. We propose that astrocyte-derived miRNA-loaded exosomes, such as miR-26a, are dysregulated in several central nervous system diseases; thus potentially controlling neuronal morphology and synaptic transmission through validated and predicted targets. Unraveling the contribution of this new signaling mechanism to the maintenance and plasticity of neuronal networks will impact our understanding on the physiology and pathophysiology of the central nervous system.

  1. The V0-ATPase mediates apical secretion of exosomes containing Hedgehog-related proteins in Caenorhabditis elegans

    Science.gov (United States)

    Liégeois, Samuel; Benedetto, Alexandre; Garnier, Jean-Marie; Schwab, Yannick; Labouesse, Michel

    2006-01-01

    Polarized intracellular trafficking in epithelia is critical in development, immunity, and physiology to deliver morphogens, defensins, or ion pumps to the appropriate membrane domain. The mechanisms that control apical trafficking remain poorly defined. Using Caenorhabditis elegans, we characterize a novel apical secretion pathway involving multivesicularbodies and the release of exosomes at the apical plasma membrane. By means of two different genetic approaches, we show that the membrane-bound V0 sector of the vacuolar H+-ATPase (V-ATPase) acts in this pathway, independent of its contribution to the V-ATPase proton pump activity. Specifically, we identified mutations in the V0 “a” subunit VHA-5 that affect either the V0-specific function or the V0+V1 function of the V-ATPase. These mutations allowed us to establish that the V0 sector mediates secretion of Hedgehog-related proteins. Our data raise the possibility that the V0 sector mediates exosome and morphogen release in mammals. PMID:16785323

  2. The biology and function of exosomes in cancer

    OpenAIRE

    Kalluri, Raghu

    2016-01-01

    Humans circulate quadrillions of exosomes at all times. Exosomes are a class of extracellular vesicles released by all cells, with a size range of 40?150 nm and a lipid bilayer membrane. Exosomes contain DNA, RNA, and proteins. Exosomes likely remove excess and/or unnecessary constituents from the cells, functioning like garbage bags, although their precise physiological role remains unknown. Additionally, exosomes may mediate specific cell-to-cell communication and activate signaling pathway...

  3. Cell to Cell Signalling via Exosomes Through esRNA

    OpenAIRE

    Lotvall, Jan; Valadi, Hadi

    2007-01-01

    Exosomes are small vesicles of endosomal origin that can be released by many different cells to the microenvironment. Exosomes have been shown to participate in the immune system, by mediating antigen presentation. We have recently shown the presence of both mRNA and microRNA in exosomes, specifically in exosomes derived from mast cells. This RNA can be transferred between one mast cell to another, most likely through fusion of the exosome to the recipient cell membrane. The delivered RNA is ...

  4. MicroRNAs transported by exosomes in body fluids as mediators of intercellular communication in cancer

    Directory of Open Access Journals (Sweden)

    Salido-Guadarrama I

    2014-07-01

    Full Text Available Iván Salido-Guadarrama,1 Sandra Romero-Cordoba,1 Oscar Peralta-Zaragoza,2 Alfredo Hidalgo-Miranda,1 Mauricio Rodríguez-Dorantes1 1Oncogenomics Laboratory, National Institute of Genomics Medicine, Mexico City, Mexico; 2Direction of Chronic Infections and Cancer, Research Center in Infectious Diseases, National Institute of Public Health, Cuernavaca, Morelos, Mexico Abstract: Cancer-cell communication is an important and complex process, achieved through a diversity of mechanisms that allows tumor cells to mold and influence their environment. In recent years, evidence has accumulated indicating that cells communicate via the release and delivery of microRNAs (miRNAs packed into tumor-released (TR exosomes. Understanding the role and mode of action of miRNAs from TR exosomes is of paramount importance in the field of cancer biomarker discovery and for the development of new biomedical applications for cancer therapeutics. In this review, we focus on miRNAs secreted via TR exosomes, which by acting in a paracrine or endocrine manner, facilitate a diversity of signaling mechanisms between cancer cells. We address their contribution as signaling molecules, to the establishment, maintenance, and enhancement of the tumor microenvironment and the metastatic niche in cancer. Finally, we address the potential role of these molecules as biomarkers in cancer diagnosis and prognosis and their impact as a biomedical tool in cancer therapeutics. Keywords: tumor cells, multivesicular bodies, interference RNA, biomarkers and therapeutics

  5. SUPERKILLER Complex Components Are Required for the RNA Exosome-Mediated Control of Cuticular Wax Biosynthesis in Arabidopsis Inflorescence Stems1[OPEN

    Science.gov (United States)

    Zhao, Lifang; Kunst, Ljerka

    2016-01-01

    ECERIFERUM7 (CER7)/AtRRP45B core subunit of the exosome, the main cellular 3′-to-5′ exoribonuclease, is a positive regulator of cuticular wax biosynthesis in Arabidopsis (Arabidopsis thaliana) inflorescence stems. CER7-dependent exosome activity determines stem wax load by controlling transcript levels of the wax-related gene CER3. Characterization of the second-site suppressors of the cer7 mutant revealed that small interfering RNAs (siRNAs) are direct effectors of CER3 expression. To explore the relationship between the exosome and posttranscriptional gene silencing (PTGS) in regulating CER3 transcript levels, we investigated two additional suppressor mutants, wax restorer1 (war1) and war7. We show that WAR1 and WAR7 encode Arabidopsis SUPERKILLER3 (AtSKI3) and AtSKI2, respectively, components of the SKI complex that associates with the exosome during cytoplasmic 3′-to-5′ RNA degradation, and that CER7-dependent regulation of wax biosynthesis also requires participation of AtSKI8. Our study further reveals that it is the impairment of the exosome-mediated 3′-5′ decay of CER3 transcript in the cer7 mutant that triggers extensive production of siRNAs and efficient PTGS of CER3. This identifies PTGS as a general mechanism for eliminating highly abundant endogenous transcripts that is activated when 3′-to-5′ mRNA turnover by the exosome is disrupted. Diminished efficiency of PTGS in ski mutants compared with cer7, as evidenced by lower accumulation of CER3-related siRNAs, suggests that reduced amounts of CER3 transcript are available for siRNA synthesis, possibly because CER3 mRNA that does not interact with SKI is degraded by 5′-to-3′ XRN4 exoribonuclease. PMID:27208312

  6. Characterization of human thymic exosomes.

    Science.gov (United States)

    Skogberg, Gabriel; Gudmundsdottir, Judith; van der Post, Sjoerd; Sandström, Kerstin; Bruhn, Sören; Benson, Mikael; Mincheva-Nilsson, Lucia; Baranov, Vladimir; Telemo, Esbjörn; Ekwall, Olov

    2013-01-01

    Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.

  7. Characterization of human thymic exosomes.

    Directory of Open Access Journals (Sweden)

    Gabriel Skogberg

    Full Text Available Exosomes are nanosized membrane-bound vesicles that are released by various cell types and are capable of carrying proteins, lipids and RNAs which can be delivered to recipient cells. Exosomes play a role in intercellular communication and have been described to mediate immunologic information. In this article we report the first isolation and characterization of exosomes from human thymic tissue. Using electron microscopy, particle size determination, density gradient measurement, flow cytometry, proteomic analysis and microRNA profiling we describe the morphology, size, density, protein composition and microRNA content of human thymic exosomes. The thymic exosomes share characteristics with previously described exosomes such as antigen presentation molecules, but they also exhibit thymus specific features regarding surface markers, protein content and microRNA profile. Interestingly, thymic exosomes carry proteins that have a tissue restricted expression in the periphery which may suggest a role in T cell selection and the induction of central tolerance. We speculate that thymic exosomes may provide the means for intercellular information exchange necessary for negative selection and regulatory T cell formation of the developing thymocytes within the human thymic medulla.

  8. Plasma Exosomes From HLA-Sensitized Kidney Transplant Recipients Contain mRNA Transcripts Which Predict Development of Antibody-Mediated Rejection.

    Science.gov (United States)

    Zhang, Hao; Huang, Edmund; Kahwaji, Joseph; Nast, Cynthia C; Li, Ping; Mirocha, James; Thomas, David L; Ge, Shili; Vo, Ashley A; Jordan, Stanley C; Toyoda, Mieko

    2017-10-01

    Sensitization to HLA remains a significant immunologic barrier to successful transplantation. Identifying immune mechanisms responsible for antibody-mediated rejection (AMR) is an important goal. Here, we explored the possibility of predicting the risk for AMR by measuring mRNA transcripts of AMR-associated genes in plasma exosomes from kidney transplant patients. Total RNA was extracted from exosomes purified from 152 ethylenediaminetetraacetic acid-plasma samples of 64 patients (18 AMR, 8 cell-mediated rejection [CMR], 38 no rejection in desensitized [DES] and non-DES control groups) for reverse transcription into cDNA, preamplification and then real time quantitative polymerase chain reaction (qPCR) for 21 candidate genes. The mRNA transcript levels of each gene were calculated. Comparisons were made among 4 patient groups for each gene and also for a gene combination score based on selected genes. Among 21 candidate genes, we identified multiple genes (gp130, CCL4, TNFα, SH2D1B, CAV1, atypical chemokine receptor 1 [duffy blood group]) whose mRNA transcript levels in plasma exosomes significantly increased among AMR compared with CMR and/or control patients. A gene combination score calculated from 4 genes of gp130, SH2D1B, TNFα, and CCL4 was significantly higher in the AMR than the CMR (P exosomes may contain information indicating clinical conditions of kidney transplant patients. mRNA transcript profiles based on gp130, SH2D1B, TNFα, and CCL4 in plasma exosomes may be used to predict on-going and/or imminent AMR.

  9. HPV-E7 Delivered by Engineered Exosomes Elicits a Protective CD8+ T Cell-Mediated Immune Response

    Directory of Open Access Journals (Sweden)

    Paola Di Bonito

    2015-03-01

    Full Text Available We developed an innovative strategy to induce a cytotoxic T cell (CTL immune response against protein antigens of choice. It relies on the production of exosomes, i.e., nanovesicles spontaneously released by all cell types. We engineered the upload of huge amounts of protein antigens upon fusion with an anchoring protein (i.e., HIV-1 Nefmut, which is an inactive protein incorporating in exosomes at high levels also when fused with foreign proteins. We compared the immunogenicity of engineered exosomes uploading human papillomavirus (HPV-E7 with that of lentiviral virus-like particles (VLPs incorporating equivalent amounts of the same antigen. These exosomes, whose limiting membrane was decorated with VSV-G, i.e., an envelope protein inducing pH-dependent endosomal fusion, proved to be as immunogenic as the cognate VLPs. It is noteworthy that the immunogenicity of the engineered exosomes remained unaltered in the absence of VSV-G. Most important, we provide evidence that the inoculation in mouse of exosomes uploading HPV-E7 induces production of anti-HPV E7 CTLs, blocks the growth of syngeneic tumor cells inoculated after immunization, and controls the development of tumor cells inoculated before the exosome challenge. These results represent the proof-of-concept about both feasibility and efficacy of the Nefmut-based exosome platform for the induction of CD8+ T cell immunity.

  10. Exosomes: promising sacks for treating ischemic heart disease?

    Science.gov (United States)

    Chen, Gui-Hao; Xu, Jun; Yang, Yue-Jin

    2017-09-01

    Ischemic heart disease(IHD) is the leading cause of death worldwide. Despite the development of continuously improving therapeutic strategies, morbidity and mortality of patients with IHD remain relatively high. Exosomes are a subpopulation of vesicles that are universally recognized as major mediators in intercellular communication. Numerous preclinical studies have shown that these tiny vesicles were protective in IHD, through such actions as alleviating myocardial ischemia-reperfusion injury, promoting angiogenesis, inhibiting fibrosis, and facilitating cardiac regeneration. Our review focused on these beneficial exosome-mediated processes. In addition, we discuss in detail how to fully exploit the therapeutic potentials of exosomes in the field of IHD. Topics include identifying robust sources of exosomes, loading protective agents into exosomes, developing heart-specific exosomes, optimizing isolation methods, and translating the cardioprotective effects of exosomes into clinical practice. Finally, both the advantages and disadvantages of utilizing exosomes in clinical settings are addressed. Copyright © 2017 the American Physiological Society.

  11. Exosomes: Some approaches to cancer diagnosis and therapy

    Science.gov (United States)

    Shtam, T.; Samsonov, R.; Kamyshinsky, R.; Pantina, R.; Verlov, N.; Vasiliev, A.; Konevega, A. L.; Malek, A. V.

    2017-09-01

    Exosomes are membrane-bound, intercellular communication shuttle vesicles that are defined by their endocytic origin and size range of 30-120 nm. Secreted by nearly all mammalian cell types and present in bodily fluids, exosomes confer messages between cells, by transporting functionally relevant proteins, nucleic acids, and lipids. The capability of tumor exosomes to house tumorigenic information and induce cellular responses that promote disease pathogenesis make tumor exosomes an attractive tool in identifying cancer biomarkers and exploiting exosomes for therapy. In this paper, we sum up our previous findings to utilize exosomes as biomarkers for early detection, diagnosis and therapy selection of prostate and thyroid cancer and present our results on exosomes in colon cancer. Some of plasma exosomal miRNAs showed their potential as diagnostic markers for colon cancer. All together, the data suggested the potentials of circulating exosomal miRNAs as liquid biopsy markers for cancer. Here we also present the possibilities of delivering therapeutic molecules by exosomes. Previously, we had demonstrated the potential of exosome-mediated siRNA delivery. Here, we present the possibility of carrying the exogenous p53 protein by exosomes in vitro.

  12. The biology and function of exosomes in cancer

    Science.gov (United States)

    Kalluri, Raghu

    2016-01-01

    Humans circulate quadrillions of exosomes at all times. Exosomes are a class of extracellular vesicles released by all cells, with a size range of 40–150 nm and a lipid bilayer membrane. Exosomes contain DNA, RNA, and proteins. Exosomes likely remove excess and/or unnecessary constituents from the cells, functioning like garbage bags, although their precise physiological role remains unknown. Additionally, exosomes may mediate specific cell-to-cell communication and activate signaling pathways in cells they fuse or interact with. Exosomes are detected in the tumor microenvironment, and emerging evidence suggests that they play a role in facilitating tumorigenesis by regulating angiogenesis, immunity, and metastasis. Circulating exosomes can be used as liquid biopsies and noninvasive biomarkers for early detection, diagnosis, and treatment of cancer patients. PMID:27035812

  13. Using exosomes, naturally-equipped nanocarriers, for drug delivery

    Science.gov (United States)

    Batrakova, Elena V.; Kim, Myung Soo

    2015-01-01

    Exosomes offer distinct advantages that uniquely position them as highly effective drug carriers. Comprised of cellular membranes with multiple adhesive proteins on their surface, exosomes are known to specialize in cell–cell communications and provide an exclusive approach for the delivery of various therapeutic agents to target cells. In addition, exosomes can be amended through their parental cells to express a targeting moiety on their surface, or supplemented with desired biological activity. Development and validation of exosome-based drug delivery systems are the focus of this review. Different techniques of exosome isolation, characterization, drug loading, and applications in experimental disease models and clinic are discussed. Exosome-based drug formulations may be applied to a wide variety of disorders such as cancer, various infectious, cardiovascular, and neuro-degenerative disorders. Overall, exosomes combine benefits of both synthetic nanocarriers and cell-mediated drug delivery systems while avoiding their limitations. PMID:26241750

  14. Epidermal Growth Factor Receptor in Prostate Cancer Derived Exosomes.

    Directory of Open Access Journals (Sweden)

    Geetanjali Kharmate

    Full Text Available Exosomes proteins and microRNAs have gained much attention as diagnostic tools and biomarker potential in various malignancies including prostate cancer (PCa. However, the role of exosomes and membrane-associated receptors, particularly epidermal growth factor receptor (EGFR as mediators of cell proliferation and invasion in PCa progression remains unexplored. EGFR is frequently overexpressed and has been associated with aggressive forms of PCa. While PCa cells and tissues express EGFR, it is unknown whether exosomes derived from PCa cells or PCa patient serum contains EGFR. The aim of this study was to detect and characterize EGFR in exosomes derived from PCa cells, LNCaP xenograft and PCa patient serum. Exosomes were isolated from conditioned media of different PCa cell lines; LNCaP xenograft serum as well as patient plasma/serum by differential centrifugation and ultracentrifugation on a sucrose density gradient. Exosomes were confirmed by electron microscopy, expression of exosomal markers and NanoSight™ analysis. EGFR expression was determined by western blot analysis and ELISA. This study demonstrates that exosomes may easily be derived from PCa cell lines, serum obtained from PCa xenograft bearing mice and clinical samples derived from PCa patients. Presence of exosomal EGFR in PCa patient exosomes may present a novel approach for measuring of the disease state. Our work will allow to build on this finding for future understanding of PCa exosomes and their potential role in PCa progression and as minimal invasive biomarkers for PCa.

  15. Secretion modification region-derived peptide blocks exosome release and mediates cell cycle arrest in breast cancer cells.

    Science.gov (United States)

    Huang, Ming-Bo; Gonzalez, Ruben R; Lillard, James; Bond, Vincent C

    2017-02-14

    Discovery and development of a novel anticancer PEG-SMR-Clu peptide to prevent breast cancer metastasis. How breast cancer cells and primary mammary epithelial cells interact and communicate with each other to promote tumorigenesis and how to prevent tumor metastasis has long been a concern of researchers. Cancer cells secrete exosomes containing proteins and RNA. These factors can influence tumor development by directly targeting cancer cells and tumor stroma. In this study, we determined the effects of a peptide as an inhibitor of exosome secretion on breast tumors. We developed a peptide derived from the Secretion Modification Region (SMR) of HIV-1 Nef protein that was modified with PEG on the N-terminus and with a Clusterin (Clu)-binding peptide on the C-terminus. Attachment of PEG to the SMR peptide, termed PEGylation, offers improved water solubility and stability as well as reduced clearance through the kidneys, leading to a longer circulation time. The 12-mer Clu-binding peptide plays multiple roles in tumor development and metastasis. The Clu peptide can be detected by antibody in vivo, thus it has the potential to be used to monitor tumor status and treatment efficacy in animal studies and eventually in cancer patients. PEG-SMRwt-Clu and PEG-SMRwt peptides inhibited the growth of both of MCF-7 (estrogen responsive, ER+) and MDA-MD-231 (estrogen non-responsive, ER-) human breast cancer cells in a dose and time-dependent manner, without inducing cytotoxic effects. The SMRwt peptide, combined with paclitaxel, induced G2/M phase cell cycle arrest on MCF-7 and MDA-MB-231 cells but did not promote apoptosis. PEG-SMRwt-Clu peptide treatment blocked exosome release from both MCF-7 and MDA-MB-231 cells. This effect was blocked by knockdown of the chaperone protein mortalin by either antibody or siRNA. MCF-7 and MDA-MB-231 breast tumor cells were treated with PEG-SMR-Clu peptide alone and in combination with paclitaxel and cisplatin. Cell proliferation and viabilty

  16. Tumour exosomes from cells harbouring PTPRZ1-MET fusion contribute to a malignant phenotype and temozolomide chemoresistance in glioblastoma.

    Science.gov (United States)

    Zeng, A-L; Yan, W; Liu, Y-W; Wang, Z; Hu, Q; Nie, E; Zhou, X; Li, R; Wang, X-F; Jiang, T; You, Y-P

    2017-09-21

    Exosomes are carriers of pro-tumorigenic factors that participate in glioblastoma (GBM) progression, and many fusion genes are strong driver mutations in neoplasia and are involved in tumorigenesis. However, the ability of fusion genes to be transduced by exosomes is unknown. We characterized exosomes from GBM cells harbouring and not harbouring PTPRZ1-MET fusion (ZM fusion). We also determined the effect of the exosomes from ZM fusion cells (ZM exosomes) on pro-oncogenic secretions and showed that ZM exosomes are internalized by the recipient cells. In addition, we studied the effect of ZM exosome-mediated intercellular communication in the GBM microenvironment. MET proto-oncogene expression was higher in ZM exosomes. Moreover, phosphorylated MET was detected only in ZM exosomes and not in exosomes released by non-ZM fusion GBM cells. ZM exosomes transferred to non-ZM fusion GBM cells and normal human astrocytes altered gene expression and induced epithelial-mesenchymal transition. The uptake of ZM exosomes also induced an exosome-dependent phenotype defined by GBM cell migration and invasion, neurosphere growth and angiogenesis. In addition, ZM exosomes conferred temozolomide resistance to the GBM cells, and exosome-derived ZM fusion network proteins targeted multiple pro-oncogenic effectors in recipient cells within the GBM microenvironment. Our findings show that exosomes mediate the aggressive character of GBM and demonstrate the role of ZM fusion in the exacerbation of this effect. These findings have possible implications for the foundation of gene fusion-based therapy for managing GBM.

  17. The human cap-binding complex is functionally connected to the nuclear RNA exosome

    DEFF Research Database (Denmark)

    Andersen, Peter Refsing; Domanski, Michal; Kristiansen, Maiken S

    2013-01-01

    Nuclear processing and quality control of eukaryotic RNA is mediated by the RNA exosome, which is regulated by accessory factors. However, the mechanism of exosome recruitment to its ribonucleoprotein (RNP) targets remains poorly understood. Here we report a physical link between the human exosome...... of combinatorial depletion of CBCN and exosome components underscore the functional relevance of CBC-exosome bridging at the level of target RNA. Specifically, CBCA suppresses read-through products of several RNA families by promoting their transcriptional termination. We suggest that the RNP 5' cap links...... transcription termination to exosomal RNA degradation through CBCN....

  18. Placental exosomes in normal and complicated pregnancy.

    Science.gov (United States)

    Mitchell, Murray D; Peiris, Hassendrini N; Kobayashi, Miharu; Koh, Yong Q; Duncombe, Gregory; Illanes, Sebastian E; Rice, Gregory E; Salomon, Carlos

    2015-10-01

    While there is considerable contemporary interest in elucidating the role of placenta-derived extracellular vesicles in normal and complicated pregnancies and their utility as biomarkers and therapeutic interventions, progress in the field is hindered by a lack of standardized extracellular vesicle taxonomy and isolation protocols. The term "extracellular vesicle" is nonspecific and refers to all membrane-bound vesicles from nanometer to micrometer diameters and of different biogenic origins. To meaningfully ascribe biological function and/or diagnostic and therapeutic utility to extracellular vesicles, and in particular exosomes, greater specificity and vesicle characterization is required. The current literature relating to exosome biology must be interpreted in this context. Exosomes are a subtype of extracellular vesicle that are specifically defined by an endosomal biogenesis and particle size (40-120 nm) and density (1.13-1.19 g/mL(-1)). Exosomes are specifically package with signaling molecules (including protein, messenger RNA, microRNA, and noncoding RNA) and are released by exocytosis into biofluid compartments. Exosomes regulate the activity of both proximal and distal target cells, including translational activity, angiogenesis, proliferation, metabolism, and apoptosis. As such, exosomal signaling represents an integral pathway mediating intercellular communication. During pregnancy, the placenta releases exosomes into the maternal circulation from as early as 6 weeks of gestation. Release is regulated by factors that include both oxygen tension and glucose concentration and correlates with placental mass and perfusion. The concentration of placenta-derived exosomes in maternal plasma increases progressively during gestation. Exosomes isolated from maternal plasma are bioactive in vitro and are incorporated into target cells by endocytosis. While the functional significance of placental exosomes in pregnancy remains to be fully elucidated, available

  19. The emerging roles of exosomes in tumor-stroma interaction.

    Science.gov (United States)

    Fu, Hailong; Yang, Huan; Zhang, Xu; Xu, Wenrong

    2016-09-01

    The tumor-stroma interaction is critical for the development and progression of cancer. Cancer-associated fibroblasts (CAFs), one of the major components of the tumor stroma, can promote tumor growth and metastasis. Exosomes are secreted microvesicles that mediate cell-to-cell communication. Exosomal contents, including proteins, nucleic acids, and lipids, can be shuttled from donor cells to target cells. Recent studies suggest that exosomes play important roles in the tumor-stroma interaction. Herein, we review the multifaceted roles of exosomes in the tumor-stroma interaction and the underlying molecular mechanisms. Literature search for all relevant publications was performed on PubMed databases. The keywords of exosomes, tumor, stroma, CAFs, mesenchymal stem cells (MSCs) and other closely related terms were used for searching. Tumor cell-derived exosomes induce the differentiation of fibroblasts and MSCs into CAFs. In turn, exosomes secreted by CAFs promote tumor growth, metastasis, and drug resistance through distinct mechanisms. Moreover, exosomes from stromal cells can be used as therapeutic vehicles for the delivery of anticancer drugs. Tumor cells communicate with CAFs through exosomes, which establishes a bidirectional cross talk to promote tumor growth, metastasis, and drug resistance. Targeting exosomes in tumor-stroma interaction may have important implications for anticancer therapy.

  20. BM mesenchymal stromal cell-derived exosomes facilitate multiple myeloma progression

    National Research Council Canada - National Science Library

    Roccaro, Aldo M; Sacco, Antonio; Maiso, Patricia; Azab, Abdel Kareem; Tai, Yu-Tzu; Reagan, Michaela; Azab, Feda; Flores, Ludmila M; Campigotto, Federico; Weller, Edie; Anderson, Kenneth C; Scadden, David T; Ghobrial, Irene M

    2013-01-01

    .... Cell-cell communication is mediated by exosomes. In this study, we showed that MM BM-MSCs release exosomes that are transferred to MM cells, thereby resulting in modulation of tumor growth in vivo. Exosomal microRNA (miR...

  1. Exosomes as agents of change in the cardiovascular system.

    Science.gov (United States)

    Poe, A J; Knowlton, A A

    2017-10-01

    Exosomes have an evolving role in paracrine and autocrine signaling, which is enhanced because these lipid vesicles are quite stable and can deliver miRNA, DNA, protein and other molecules to cells throughout the body. Most cell types release exosomes, and exosomes are found in all biological fluids, making them accessible biomarkers. Significantly, exosomes can carry a biologically potent cargo, which can alter the phenotype of recipient cells. In the cardiovascular system exosomes have been primarily studied for their role in mediating the beneficial effects of mesenchymal stem cells after myocardial injury. Exosomes released by cardiac cells in disease states, such as myocardial ischemia, can potentially have important pathophysiologic effects on other cardiac cells as well as on distant organs. Published by Elsevier Ltd.

  2. Exosomes and Cardiovascular Protection.

    Science.gov (United States)

    Davidson, Sean M; Takov, Kaloyan; Yellon, Derek M

    2017-02-01

    Most, if not all, cells of the cardiovascular system secrete small, lipid bilayer vesicles called exosomes. Despite technical challenges in their purification and analysis, exosomes from various sources have been shown to be powerfully cardioprotective. Indeed, it is possible that much of the so-called "paracrine" benefit in cardiovascular function obtained by stem cell therapy can be replicated by the injection of exosomes produced by stem cells. However, exosomes purified from plasma appear to be just as capable of activating cardioprotective pathways. We discuss the potential roles of endogenous exosomes in the cardiovascular system, how this is perturbed in cardiovascular disease, and evaluate their potential as therapeutic agents to protect the heart.

  3. Exosomes: Mechanisms of Uptake

    Directory of Open Access Journals (Sweden)

    Kelly J. McKelvey

    2015-07-01

    Full Text Available Exosomes are 30–100 nm microvesicles which contain complex cellular signals of RNA, protein and lipids. Because of this, exosomes are implicated as having limitless therapeutic potential for the treatment of cancer, pregnancy complications, infections, and autoimmune diseases. To date we know a considerable amount about exosome biogenesis and secretion, but there is a paucity of data regarding the uptake of exosomes by immune and non-immune cell types (e.g., cancer cells and the internal signalling pathways by which these exosomes elicit a cellular response. Answering these questions is of paramount importance.

  4. Exosomes: Mechanisms of Uptake

    Directory of Open Access Journals (Sweden)

    Kelly J. McKelvey

    2015-07-01

    Full Text Available Exosomes are 30–100 nm microvesicles which contain complex cellular signals of RNA, protein and lipids. Because of this, exosomes are implicated as having limitless therapeutic potential for the treatment of cancer, pregnancy complications, infections, and autoimmune diseases. To date we know a considerable amount about exosome biogenesis and secretion, but there is a paucity of data regarding the uptake of exosomes by immune and non- immune cell types (e.g., cancer cells and the internal signalling pathways by which these exosomes elicit a cellular response. Answering these questions is of para‐ mount importance.

  5. Low-Intensity Ultrasound-Induced Anti-inflammatory Effects Are Mediated by Several New Mechanisms Including Gene Induction, Immunosuppressor Cell Promotion, and Enhancement of Exosome Biogenesis and Docking

    Directory of Open Access Journals (Sweden)

    Qian Yang

    2017-10-01

    Full Text Available Background: Low-intensity ultrasound (LIUS was shown to be beneficial in mitigating inflammation and facilitating tissue repair in various pathologies. Determination of the molecular mechanisms underlying the anti-inflammatory effects of LIUS allows to optimize this technique as a therapy for the treatment of malignancies and aseptic inflammatory disorders.Methods: We conducted cutting-edge database mining approaches to determine the anti-inflammatory mechanisms exerted by LIUS.Results: Our data revealed following interesting findings: (1 LIUS anti-inflammatory effects are mediated by upregulating anti-inflammatory gene expression; (2 LIUS induces the upregulation of the markers and master regulators of immunosuppressor cells including MDSCs (myeloid-derived suppressor cells, MSCs (mesenchymal stem cells, B1-B cells and Treg (regulatory T cells; (3 LIUS not only can be used as a therapeutic approach to deliver drugs packed in various structures such as nanobeads, nanospheres, polymer microspheres, and lipidosomes, but also can make use of natural membrane vesicles as small as exosomes derived from immunosuppressor cells as a novel mechanism to fulfill its anti-inflammatory effects; (4 LIUS upregulates the expression of extracellular vesicle/exosome biogenesis mediators and docking mediators; (5 Exosome-carried anti-inflammatory cytokines and anti-inflammatory microRNAs inhibit inflammation of target cells via multiple shared and specific pathways, suggesting exosome-mediated anti-inflammatory effect of LIUS feasible; and (6 LIUS-mediated physical effects on tissues may activate specific cellular sensors that activate downstream transcription factors and signaling pathways.Conclusions: Our results have provided novel insights into the mechanisms underlying anti-inflammatory effects of LIUS, and have provided guidance for the development of future novel therapeutic LIUS for cancers, inflammatory disorders, tissue regeneration and tissue repair.

  6. HIV-1 Nef Signaling in Intestinal Mucosa Epithelium Suggests the Existence of an Active Inter-kingdom Crosstalk Mediated by Exosomes

    Directory of Open Access Journals (Sweden)

    Cristina Felli

    2017-06-01

    Full Text Available The human intestinal mucosal surface represents the first defense against pathogens and regulates the immune response through the combination of epithelial cell (EC functions and immunological factors. ECs act as sensors of luminal stimuli and interact with the immune cells through signal–transduction pathways, thus representing the first barrier that HIV-1 virus encounters during infection. In particular, the HIV-1 Nef protein plays a crucial role in viral invasion and replication. Nef is expressed early during viral infection and interacts with numerous cellular proteins as a scaffold/adaptor. Nef is localized primarily to cellular membranes and affects several signaling cascades in infected cells modulating the expression of cell surface receptors critical for HIV-1 infection and transmission, also accompanied by the production of specific cytokines and progressive depletion of CD4+ T cells. At the intestinal level, Nef contributes to affect the mucosal barrier by increasing epithelial permeability, that results in the translocation of microbial antigens and consequently in immune system activation. However, the pathological role of Nef in mucosal dysfunction has not been fully elucidated. Interestingly, Nef is secreted also within exosomes and contributes to regulate the intercellular communication exploiting the vesicular trafficking machinery of the host. This can be considered as a potential inter-kingdom communication pathway between virus and humans, where viral Nef contributes to modulate and post-transcriptionally regulate the host gene expression and immune response. In this mini-review we discuss the effects of HIV-1 Nef protein on intestinal epithelium and propose the existence of an inter-kingdom communication process mediated by exosomes.

  7. Cells release subpopulations of exosomes with distinct molecular and biological properties.

    Science.gov (United States)

    Willms, Eduard; Johansson, Henrik J; Mäger, Imre; Lee, Yi; Blomberg, K Emelie M; Sadik, Mariam; Alaarg, Amr; Smith, C I Edvard; Lehtiö, Janne; El Andaloussi, Samir; Wood, Matthew J A; Vader, Pieter

    2016-03-02

    Cells release nano-sized membrane vesicles that are involved in intercellular communication by transferring biological information between cells. It is generally accepted that cells release at least three types of extracellular vesicles (EVs): apoptotic bodies, microvesicles and exosomes. While a wide range of putative biological functions have been attributed to exosomes, they are assumed to represent a homogenous population of EVs. We hypothesized the existence of subpopulations of exosomes with defined molecular compositions and biological properties. Density gradient centrifugation of isolated exosomes revealed the presence of two distinct subpopulations, differing in biophysical properties and their proteomic and RNA repertoires. Interestingly, the subpopulations mediated differential effects on the gene expression programmes in recipient cells. In conclusion, we demonstrate that cells release distinct exosome subpopulations with unique compositions that elicit differential effects on recipient cells. Further dissection of exosome heterogeneity will advance our understanding of exosomal biology in health and disease and accelerate the development of exosome-based diagnostics and therapeutics.

  8. Exosomes in Critical Illness.

    Science.gov (United States)

    Terrasini, Nora; Lionetti, Vincenzo

    2017-06-01

    Exosomes are small, cell-released vesicles (40-100 nm in size) with the potential to transfer proteins, lipids, small RNAs, messenger RNAs, or DNA between cells via interstitial fluids. Due to their role in tissue homeostasis, exosomes have emerged as a new type of therapeutic and diagnostic (theranostic) tool in the noninvasive assessment of organ response to injury or treatment and in the development of reliable organ-protective intensive therapy. Our review provides current insights into the role of exosomes in the personalized management of injury and repair responses in critical illness. Data were obtained from a PubMed search of the most recent medical literature, including the PubMed "related articles" search methodology. Articles considered include original articles, review articles and conference proceedings. A detailed review of scientific, peer-reviewed data was performed. Relevant pre-clinical and clinical studies were included and summarized. Current scientific evidence is focused on the following: 1) Frontiers in the management of critical illness; 2) Biogenesis, characterization, and function of circulating exosomes; 3) The role of exosomes in acute lung injury; 4) The role of exosomes in acute cardiac injury; 5) The role of exosomes in acute kidney injury; 6) The role of exosomes in sepsis; 7) Limitations of exosome isolation protocols; and 8) Perspectives in the theranostic use of exosomes. Circulating levels of exosomes are associated with the onset and clinical course of critical illness. Exosomes released from cells with different phenotypes exert different functions in order to protect tissue and preserve organ function. Therefore, multifunctional exosomes with combined diagnostic and therapeutic functions show great promise in terms of personalized nanomedicine for patient-specific diagnosis and treatment of critical illness.

  9. Enrichment of selective miRNAs in exosomes and delivery of exosomal miRNAs in vitro and in vivo.

    Science.gov (United States)

    Zhang, Duo; Lee, Heedoo; Zhu, Ziwen; Minhas, Jasleen K; Jin, Yang

    2017-01-01

    Exosomes are nanovesicles secreted by cells and contain various molecules including protein, lipid, and DNA/RNA. They are crucial mediators of the intercellular communication and serve as promising vehicles for drug delivery and gene therapy. Recently, accumulating evidence suggests that microRNAs (miRNAs) may serve as new and potentially powerful targets for therapeutic interventions against various human diseases. However, steadily and effectively delivering miRNA mimics or inhibitors to target cells remains a major obstacle. To enhance the efficacy of exosome-mediated delivery of miRNA molecules, it is crucial to develop a convenient and efficient method to enrich specific miRNAs or antisense oligos in isolated exosomes. Here we report a novel method to prepare specific miRNA molecule-loaded exosomes. Using a modified calcium chloride-mediated transfection method, we successfully enhanced the designated miRNA mimics or inhibitors in isolated exosomes directly, instead of transfecting their mother cells. We also compared this method with direct transfection of exosomes using electroporation. Both methods confirmed that exosomes can serve as cargos to deliver a robustly increased amount of selected miRNA mimic(s) or inhibitor(s) to the recipient cells. Delivery of these miRNA molecule enriched-exosomes subsequently results in highly efficient overexpression or deletion of the designated miRNAs in the recipient cells both in vivo and in vitro. Additionally, we confirmed that exosome-delivered miRNA mimics or inhibitors are functional in the recipient cells. Collectively, we developed a novel protocol to conveniently manipulate exosomal miRNAs with high efficiency and successfully deliver the exosomal miRNA molecules to recipient cells. Copyright © 2017 the American Physiological Society.

  10. Characteristics and Roles of Exosomes in Cardiovascular Disease.

    Science.gov (United States)

    Zhang, Yuan; Hu, Yan-Wei; Zheng, Lei; Wang, Qian

    2017-03-01

    Exosomes are nano-sized biological membrane-enclosed vesicles that contain a cell-specific cargo of proteins, lipids, and nucleic acids that are released and taken up by most cell types, thereby inducing expression and functional changes via horizontal transfer of cargos between cells. Thus, exosomes present a largely unknown "cell-to-cell" communication system, which is now increasingly being investigated for diagnostic and therapeutic use in cardiovascular disease (CVD). The purpose of this review is to summarize recent findings on the properties and roles of exosomes in a variety of physiological and pathological settings related to CVD. We focus on available information on exosome-mediated intercellular communication relevant to myocardial injury, repair, and regeneration. Finally, we address the promise of exosomes as valuable diagnostic and prognostic biomarkers, and their potential use as therapeutic tools in CVD. Exosomes remain largely unexplored for therapeutic use in the field of cardiovascular diagnosis and medicine. A more detailed characterization of cardiac exosomes shed by different components of the heart will be of fundamental importance to address specific changes in the profile of exosomal microRNAs and proteins, which will enable the clinical use of exosomes as minimally invasive diagnostic tools and vehicles for delivery of targeted therapies for CVD.

  11. Development of exosome surface display technology in living human cells.

    Science.gov (United States)

    Stickney, Zachary; Losacco, Joseph; McDevitt, Sophie; Zhang, Zhiwen; Lu, Biao

    2016-03-25

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell-cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  12. Development of exosome surface display technology in living human cells

    Energy Technology Data Exchange (ETDEWEB)

    Stickney, Zachary, E-mail: zstickney@scu.edu; Losacco, Joseph, E-mail: jlosacco@scu.edu; McDevitt, Sophie, E-mail: smmcdevitt@scu.edu; Zhang, Zhiwen, E-mail: zzhang@scu.edu; Lu, Biao, E-mail: blu2@scu.edu

    2016-03-25

    Surface display technology is an emerging key player in presenting functional proteins for targeted drug delivery and therapy. Although a number of technologies exist, a desirable mammalian surface display system is lacking. Exosomes are extracellular vesicles that facilitate cell–cell communication and can be engineered as nano-shuttles for cell-specific delivery. In this study, we report the development of a novel exosome surface display technology by exploiting mammalian cell secreted nano-vesicles and their trans-membrane protein tetraspanins. By constructing a set of fluorescent reporters for both the inner and outer surface display on exosomes at two selected sites of tetraspanins, we demonstrated the successful exosomal display via gene transfection and monitoring fluorescence in vivo. We subsequently validated our system by demonstrating the expected intracellular partitioning of reporter protein into sub-cellular compartments and secretion of exosomes from human HEK293 cells. Lastly, we established the stable engineered cells to harness the ability of this robust system for continuous production, secretion, and uptake of displayed exosomes with minimal impact on human cell biology. In sum, our work paved the way for potential applications of exosome, including exosome tracking and imaging, targeted drug delivery, as well as exosome-mediated vaccine and therapy.

  13. Cancer Exosomes Perform Cell-Independent MicroRNA Biogenesis and Promote Tumorigenesis

    Science.gov (United States)

    Melo, Sonia A.; Sugimoto, Hikaru; O’Connell, Joyce T.; Kato, Noritoshi; Villanueva, Alberto; Vidal, August; Qiu, Le; Vitkin, Edward; Perelman, Lev T.; Melo, Carlos A.; Lucci, Anthony; Ivan, Cristina; Calin, George A.; Kalluri, Raghu

    2014-01-01

    SUMMARY Exosomes are secreted by all cell types and contain proteins and nucleic acids. Here, we report that breast cancer associated exosomes contain microRNAs (miRNAs) associated with the RISC Loading Complex (RLC) and display cell-independent capacity to process precursor microRNAs (pre-miRNAs) into mature miRNAs. Pre-miRNAs, along with Dicer, AGO2, and TRBP, are present in exosomes of cancer cells. CD43 mediates the accumulation of Dicer specifically in cancer exosomes. Cancer exosomes mediate an efficient and rapid silencing of mRNAs to reprogram the target cell transcriptome. Exosomes derived from cells and sera of patients with breast cancer instigate non-tumorigenic epithelial cells to form tumors in a Dicer-dependent manner. These findings offer opportunities for the development of exosomes based biomarkers and therapies. PMID:25446899

  14. Inflammasome-Derived Exosomes Activate NF-κB Signaling in Macrophages.

    Science.gov (United States)

    Zhang, Yuehui; Liu, Fangbing; Yuan, Yanzhi; Jin, Chaozhi; Chang, Cheng; Zhu, Yunping; Zhang, Xiuyuan; Tian, Chunyan; He, Fuchu; Wang, Jian

    2017-01-06

    Exosomes are secreted small vesicles that mediate various biological processes, such as tumorigenesis and immune response. However, whether the inflammasome signaling leads to the change of constituent of exosomes and its roles in immune response remains to be determined. We isolated the exosomes from macrophages with treatment of mock, endotoxin, or endotoxin/nigericin. A label-free quantification method by MS/MS was used to identify the components of exosomes. In total, 2331 proteins were identified and 513 proteins were exclusively detected in exosomes with endotoxin and nigericin treatment. The differentially expressed proteins were classified by Gene Ontology and KEGG pathways. The immune response-related proteins and signaling pathways were specifically enriched in inflammasome-derived exosomes. Moreover, we treated macrophages with the exosomes from different stimulation. We found that inflammasome-derived exosomes directly activate NF-κB signaling pathway, while the control or endotoxin-derived exosomes have no effect. The inflammatory signaling was amplified in neighbor cells in an exosome-dependent way. The inflammasome-derived exosomes might be used to augment the immune response in disease treatment, and preventing the transfer of these exosomes might ameliorate autoimmune diseases.

  15. Experimental, Systems, and Computational Approaches to Understanding the MicroRNA-Mediated Reparative Potential of Cardiac Progenitor Cell-Derived Exosomes From Pediatric Patients.

    Science.gov (United States)

    Agarwal, Udit; George, Alex; Bhutani, Srishti; Ghosh-Choudhary, Shohini; Maxwell, Joshua T; Brown, Milton E; Mehta, Yash; Platt, Manu O; Liang, Yaxuan; Sahoo, Susmita; Davis, Michael E

    2017-02-17

    Studies have demonstrated that exosomes can repair cardiac tissue post-myocardial infarction and recapitulate the benefits of cellular therapy. We evaluated the role of donor age and hypoxia of human pediatric cardiac progenitor cell (CPC)-derived exosomes in a rat model of ischemia-reperfusion injury. Human CPCs from the right atrial appendages from children of different ages undergoing cardiac surgery for congenital heart defects were isolated and cultured under hypoxic or normoxic conditions. Exosomes were isolated from the culture-conditioned media and delivered to athymic rats after ischemia-reperfusion injury. Echocardiography at day 3 post-myocardial infarction suggested statistically improved function in neonatal hypoxic and neonatal normoxic groups compared with saline-treated controls. At 28 days post-myocardial infarction, exosomes derived from neonatal normoxia, neonatal hypoxia, infant hypoxia, and child hypoxia significantly improved cardiac function compared with those from saline-treated controls. Staining showed decreased fibrosis and improved angiogenesis in hypoxic groups compared with controls. Finally, using sequencing data, a computational model was generated to link microRNA levels to specific outcomes. CPC exosomes derived from neonates improved cardiac function independent of culture oxygen levels, whereas CPC exosomes from older children were not reparative unless subjected to hypoxic conditions. Cardiac functional improvements were associated with increased angiogenesis, reduced fibrosis, and improved hypertrophy, resulting in improved cardiac function; however, mechanisms for normoxic neonatal CPC exosomes improved function independent of those mechanisms. This is the first study of its kind demonstrating that donor age and oxygen content in the microenvironment significantly alter the efficacy of human CPC-derived exosomes. © 2016 American Heart Association, Inc.

  16. The roles and implications of exosomes in sarcoma

    Science.gov (United States)

    Min, Li; Shen, Jacson; Tu, Chongqi; Hornicek, Francis; Duan, Zhenfeng

    2016-01-01

    Better diagnostic biomarkers and therapeutic options are still necessary for patients with sarcomas due to the current limitations of diagnosis and treatment. Exosomes are small extracellular membrane vesicles that are released by various cells and are found in most body fluids. Tumor-derived exosomes have been proven to mediate tumorigenesis, intercellular communication, microenvironment modulation, and metastasis in different cancers, including in sarcomas. Recently, exosomes have been considered as potential biomarkers for sarcoma diagnosis, prognosis, and possible targets for sarcoma therapy. Moreover, due to their specific cell-tropism and bioavailability, exosomes can also be engineered as vehicles for drug delivery. In this review, we discuss recent advances in the roles of tumor-derived exosomes in sarcoma and their potential clinical applications. PMID:27342745

  17. Therapeutic Uses of Exosomes

    Directory of Open Access Journals (Sweden)

    Zacharias E. Suntres

    2013-01-01

    Full Text Available Exosomes are membrane vesicles with a diameter of 40–100 nm that are secreted by many cell types into the extracellular milieu. Exosomes are found in cell culture supernatants and in different biological fluids and are known to be secreted by most cell types under normal and pathological conditions. Considerable research is focusing on the exploitation of exosomes in biological fluids for biomarkers in the diagnosis of disease. More recently, exosomes are being exploited for their therapeutic potential. Exosomes derived from dendritic cells, tumor cells, and malignant effusions demonstrate immunomodulatory functions and are able to present antigens to T-cells and stimulate antigen-specific T-cell responses. Exosomes have also been examined for their therapeutic potential in the treatment of infections such as toxoplasmosis, diphtheria, tuberculosis and atypical severe acute respiratory syndrome as well as autoimmune diseases. Attempts to find practical applications for exosomes continue to expand with the role of exosomes as a drug delivery system for the treatment of autoimmune/inflammatory diseases and cancers.

  18. Isolation and Characterization of CD34+ Blast-Derived Exosomes in Acute Myeloid Leukemia

    Science.gov (United States)

    Hong, Chang Sook; Muller, Laurent; Boyiadzis, Michael; Whiteside, Theresa L.

    2014-01-01

    Exosomes are membrane-bound vesicles found in all biological fluids. AML patients' plasma collected at diagnosis contains elevated exosome levels relative to normal donor (ND) plasma. The molecular profile of AML exosomes changes in the course of therapy and may serve as a measure of disease progression or response to therapy. However, plasma contains a mix of exosomes derived from various cell types. To be able to utilize blast-derived exosomes as biomarkers for AML, we have developed an immunoaffinity-based capture method utilizing magnetic microbeads coated with anti-CD34 antibody (Ab). This Ab is specific for CD34, a unique marker of AML blasts. The capture procedure was developed using CD34+ exosomes derived from Kasumi-1 AML cell culture supernatants. The capture capacity of CD34microbeads was shown to linearly correlate with the input exosomes. A 10 uL aliquot of CD34 microbeads was able to capture all of CD34+ exosomes present in 100–1,000 uL of AML plasma. The levels of immunocaptured CD34+ exosomes correlated with the percentages of CD34+ blasts in the AML patients' peripheral blood. The immunocaptured exosomes had a typical cup-shaped morphology by transmission electron microscopy, and their molecular cargo was similar to that of parental blasts. These exosomes were biologically-active. Upon co-incubation with natural killer (NK) cells, captured blast-derived exosomes down-regulated surface NKG2D expression, while non-captured exosomes reduced expression levels of NKp46. Our data provide a proof-of-principle that blast-derived exosomes can be quantitatively recovered from AML patients' plasma, their molecular profile recapitulates that of autologous blasts and they retain the ability to mediate immune suppression. These data suggest that immunocaptured blast-derived exosomes might be useful in diagnosis and/or prognosis of AML in the future. PMID:25093329

  19. Functional significance of macrophage-derived exosomes in inflammation and pain.

    Science.gov (United States)

    McDonald, Marguerite K; Tian, Yuzhen; Qureshi, Rehman A; Gormley, Michael; Ertel, Adam; Gao, Ruby; Aradillas Lopez, Enrique; Alexander, Guillermo M; Sacan, Ahmet; Fortina, Paolo; Ajit, Seena K

    2014-08-01

    Exosomes, secreted microvesicles transporting microRNAs (miRNAs), mRNAs, and proteins through bodily fluids, facilitate intercellular communication and elicit immune responses. Exosomal contents vary, depending on the source and the physiological conditions of cells, and can provide insights into how cells and systems cope with physiological perturbations. Previous analysis of circulating miRNAs in patients with complex regional pain syndrome (CRPS), a debilitating chronic pain disorder, revealed a subset of miRNAs in whole blood that are altered in the disease. To determine functional consequences of alterations in exosomal biomolecules in inflammation and pain, we investigated exosome-mediated information transfer in vitro, in a rodent model of inflammatory pain, and in exosomes from patients with CRPS. Mouse macrophage cells stimulated with lipopolysaccharides secrete exosomes containing elevated levels of cytokines and miRNAs that mediate inflammation. Transcriptome sequencing of exosomal RNA revealed global alterations in both innate and adaptive immune pathways. Exosomes from lipopolysaccharide-stimulated cells were sufficient to cause nuclear factor-κB activation in naive cells, indicating functionality in recipient cells. A single injection of exosomes attenuated thermal hyperalgesia in a murine model of inflammatory pain, suggesting an immunoprotective role for macrophage-derived exosomes. Macrophage-derived exosomes carry a protective signature that is altered when secreting cells are exposed to an inflammatory stimulus. We also show that circulating miRNAs altered in patients with complex regional pain syndrome are trafficked by exosomes. With their systemic signaling capabilities, exosomes can induce pleiotropic effects potentially mediating the multifactorial pathology underlying chronic pain, and should be explored for their therapeutic utility. Copyright © 2014 International Association for the Study of Pain. Published by Elsevier B.V. All rights

  20. Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation

    Energy Technology Data Exchange (ETDEWEB)

    Hazawa, Masaharu; Tomiyama, Kenichi; Saotome-Nakamura, Ai; Obara, Chizuka; Yasuda, Takeshi; Gotoh, Takaya; Tanaka, Izumi; Yakumaru, Haruko; Ishihara, Hiroshi; Tajima, Katsushi, E-mail: tajima@nirs.go.jp

    2014-04-18

    Highlights: • Radiation increases cellular uptake of exosomes. • Radiation induces colocalization of CD29 and CD81. • Exosomes selectively bind the CD29/CD81 complex. • Radiation increases the cellular uptake of exosomes through CD29/CD81 complex formation. - Abstract: Exosomes mediate intercellular communication, and mesenchymal stem cells (MSC) or their secreted exosomes affect a number of pathophysiologic states. Clinical applications of MSC and exosomes are increasingly anticipated. Radiation therapy is the main therapeutic tool for a number of various conditions. The cellular uptake mechanisms of exosomes and the effects of radiation on exosome–cell interactions are crucial, but they are not well understood. Here we examined the basic mechanisms and effects of radiation on exosome uptake processes in MSC. Radiation increased the cellular uptake of exosomes. Radiation markedly enhanced the initial cellular attachment to exosomes and induced the colocalization of integrin CD29 and tetraspanin CD81 on the cell surface without affecting their expression levels. Exosomes dominantly bound to the CD29/CD81 complex. Knockdown of CD29 completely inhibited the radiation-induced uptake, and additional or single knockdown of CD81 inhibited basal uptake as well as the increase in radiation-induced uptake. We also examined possible exosome uptake processes affected by radiation. Radiation-induced changes did not involve dynamin2, reactive oxygen species, or their evoked p38 mitogen-activated protein kinase-dependent endocytic or pinocytic pathways. Radiation increased the cellular uptake of exosomes through CD29/CD81 complex formation. These findings provide essential basic insights for potential therapeutic applications of exosomes or MSC in combination with radiation.

  1. Exosomes Derived from Mesenchymal Stromal Cells Promote Axonal Growth of Cortical Neurons.

    Science.gov (United States)

    Zhang, Yi; Chopp, Michael; Liu, Xian Shuang; Katakowski, Mark; Wang, Xinli; Tian, Xinchu; Wu, David; Zhang, Zheng Gang

    2017-05-01

    Treatment of brain injury with exosomes derived from mesenchymal stromal cells (MSCs) enhances neurite growth. However, the direct effect of exosomes on axonal growth and molecular mechanisms underlying exosome-enhanced neurite growth are not known. Using primary cortical neurons cultured in a microfluidic device, we found that MSC-exosomes promoted axonal growth, whereas attenuation of argonaut 2 protein, one of the primary microRNA (miRNA) machinery proteins, in MSC-exosomes abolished their effect on axonal growth. Both neuronal cell bodies and axons internalized MSC-exosomes, which was blocked by botulinum neurotoxins (BoNTs) that cleave proteins of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Moreover, tailored MSC-exosomes carrying elevated miR-17-92 cluster further enhanced axonal growth compared to native MSC-exosomes. Quantitative RT-PCR and Western blot analysis showed that the tailored MSC-exosomes increased levels of individual members of this cluster and activated the PTEN/mTOR signaling pathway in recipient neurons, respectively. Together, our data demonstrate that native MSC-exosomes promote axonal growth while the tailored MSC-exosomes can further boost this effect and that tailored exosomes can deliver their selective cargo miRNAs into and activate their target signals in recipient neurons. Neuronal internalization of MSC-exosomes is mediated by the SNARE complex. This study reveals molecular mechanisms that contribute to MSC-exosome-promoted axonal growth, which provides a potential therapeutic strategy to enhance axonal growth.

  2. Exosomes as immunotheranostic nanoparticles.

    Science.gov (United States)

    Natasha G; Gundogan, Buket; Tan, Aaron; Farhatnia, Yasmin; Wu, Woffles; Rajadas, Jayakumar; Seifalian, Alexander M

    2014-06-01

    Exosomes are small biological membrane vesicles that measure 30 to 100 nm in diameter. They are involved in a wide array of biological activities, such as cell-cell communication, signal transduction, transport of genetic materials, and modulation of immune response. Evidence indicates that they can be used as not only therapeutic agents targeted against disease but also diagnostic biomarkers for pathologic conditions. In this review, we endeavor to present exosomes as immunologic agents that can be used as pioneering cancer vaccines to prime the immune system and explicate their therapeutic and diagnostic capabilities. An extensive literature search for studies that involved the use of exosomes as immunotheranostic nanoparticles was conducted using PubMed, ISI Web of Knowledge, and Google Scholar. Clinical trials that involved exosomes were also compiled by searching the clinicaltrials.gov database. In its therapeutic facet of application, exosomes can be used as vehicles for drug or gene delivery. These biological vesicles have been found to have excellent host biodistribution and biocompatibility, issues often presented with gene delivery vehicles. Diagnostically, exosomes may prove to be useful biomarkers that are able to surpass current setbacks of modern diagnostic testing, which include invasive methods. Finally, current evidence has implied that the use of exosomes could form the basis for the development of future cell-free cancer vaccines. Exosomes have numerous functions, and their double-edged features make the scope of their clinical applications, as both a diagnostic and therapeutic tool, immense. Crown Copyright © 2014. Published by EM Inc USA. All rights reserved.

  3. Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect

    OpenAIRE

    Le, Michelle; Fernandez-Palomo, Cristian; McNeill, Fiona E.; Seymour, Colin B.; Rainbow, Andrew J.; Mothersill, Carmel E.

    2017-01-01

    OBJECTIVE: The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV) biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal. METHODS: The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells...

  4. Exosomes are released by bystander cells exposed to radiation-induced biophoton signals: Reconciling the mechanisms mediating the bystander effect

    OpenAIRE

    Le, Michelle; Fernandez-Palomo, Cristian; McNeill, Fiona E.; Seymour, Colin B.; Rainbow, Andrew J.; Mothersill, Carmel E

    2017-01-01

    Objective The objective of our study was to explore a possible molecular mechanism by which ultraviolet (UV) biophotons could elicit bystander responses in reporter cells and resolve the problem of seemingly mutually exclusive mechanisms of a physical UV signal & a soluble factor-mediated bystander signal. Methods The human colon carcinoma cell line, HCT116 p53 +/+, was directly irradiated with 0.5 Gy tritium beta particles to induce ultraviolet biophoton emission. Bystander cells were not di...

  5. Exosomes carrying immunoinhibitory proteins and their role in cancer.

    Science.gov (United States)

    Whiteside, T L

    2017-09-01

    Recent emergence of exosomes as information carriers between cells has introduced us to a new previously unknown biological communication system. Multi-directional cross-talk mediated by exosomes carrying proteins, lipids and nucleic acids between normal cells, cells harbouring a pathogen or cancer and immune cells has been instrumental in determining outcomes of physiological as well as pathological conditions. Exosomes play a key role in the broad spectrum of human diseases. In cancer, tumour-derived exosomes carry multiple immunoinhibitory signals, disable anti-tumour immune effector cells and promote tumour escape from immune control. Exosomes delivering negative signals to immune cells in cancer, viral infections, autoimmune or other diseases may interfere with therapy and influence outcome. Exosomes can activate tissue cells to produce inhibitory factors and thus can suppress the host immune responses indirectly. Exosomes also promise to be non-invasive disease biomarkers with a dual capability to provide insights into immune dysfunction as well as disease progression and outcome. © 2017 British Society for Immunology.

  6. Exosomes: Nanoparticulate tools for RNA interference and drug delivery.

    Science.gov (United States)

    Shahabipour, Fahimeh; Barati, Nastaran; Johnston, Thomas P; Derosa, Giuseppe; Maffioli, Pamela; Sahebkar, Amirhossein

    2017-07-01

    Exosomes are naturally occurring extracellular vesicles released by most mammalian cells in all body fluids. Exosomes are known as key mediators in cell-cell communication and facilitate the transfer of genetic and biochemical information between distant cells. Structurally, exosomes are composed of lipids, proteins, and also several types of RNAs which enable these vesicles to serve as important disease biomarkers. Moreover, exosomes have emerged as novel drug and gene delivery tools owing to their multiple advantages over conventional delivery systems. Recently, increasing attention has been focused on exosomes for the delivery of drugs, including therapeutic recombinant proteins, to various target tissues. Exosomes are also promising vehicles for the delivery of microRNAs and small interfering RNAs, which is usually hampered by rapid degradation of these RNAs, as well as inefficient tissue specificity of currently available delivery strategies. This review highlights the most recent accomplishments and trends in the use of exosomes for the delivery of drugs and therapeutic RNA molecules. © 2017 Wiley Periodicals, Inc.

  7. Tumor-derived exosomes and their role in cancer progression

    Science.gov (United States)

    Whiteside, Theresa L

    2017-01-01

    Tumor cells actively produce, release and utilize exosomes to promote tumor growth. Mechanisms through which tumor-derived exosomes subserve the tumor are under intense investigation. These exosomes are information carriers, conveying molecular and genetic messages from tumor cells to normal or other abnormal cells residing at close or distant sites. Tumor-derived exosomes are found in all body fluids. Upon the contact with target cells, they alter phenotypic and functional attributes of recipients, reprogramming them into active contributors to angiogenesis, thrombosis, metastasis and immunosuppression. Exosomes produced by tumors carry cargos that in part mimic contents of parent cells and are of potential interest as non-invasive biomarkers of cancer. Their role in inhibiting the host antitumor responses and in mediating drug resistance is important for cancer therapy. Tumor-derived exosomes may interfere with cancer immunotherapy, but they also could serve as adjuvants and antigenic components of antitumor vaccines. Their biological roles in cancer development or progression as well as cancer therapy suggest that tumor-derived exosomes are critical components of oncogenic transformation. PMID:27117662

  8. Exosomes and Their Therapeutic Potentials of Stem Cells

    Directory of Open Access Journals (Sweden)

    Chao Han

    2016-01-01

    Full Text Available Exosomes, a group of vesicles originating from the multivesicular bodies (MVBs, are released into the extracellular space when MVBs fuse with the plasma membrane. Numerous studies indicate that exosomes play important roles in cell-to-cell communication, and exosomes from specific cell types and conditions display multiple functions such as exerting positive effects on regeneration in many tissues. It is widely accepted that the therapeutic potential of stem cells may be mediated largely by the paracrine factors, so harnessing the paracrine effects of stem and progenitor cells without affecting these living, replicating, and potentially pluripotent cell populations is an advantage in terms of safety and complexity. Ascending evidence indicated that exosomes might be the main components of paracrine factors; thus, understanding the role of exosomes in each subtype of stem cells is far-reaching. In this review, we discuss the functions of exosomes from different types of stem cells and emphasize the therapeutic potentials of exosomes, providing an alternative way of developing strategies to cure diseases.

  9. Enhancement of Gemcitabine sensitivity in pancreatic adenocarcinoma by novel exosome-mediated delivery of the Survivin-T34A mutant

    Directory of Open Access Journals (Sweden)

    Jonathan R. Aspe

    2014-02-01

    Full Text Available Background: Current therapeutic options for advanced pancreatic cancer have been largely disappointing with modest results at best, and though adjuvant therapy remains controversial, most remain in agreement that Gemcitabine should stand as part of any combination study. The inhibitor of apoptosis (IAP protein Survivin is a key factor in maintaining apoptosis resistance, and its dominant-negative mutant (Survivin-T34A has been shown to block Survivin, inducing caspase activation and apoptosis. Methods: In this study, exosomes, collected from a melanoma cell line built to harbor a tetracycline-regulated Survivin-T34A, were plated on the pancreatic adenocarcinoma (MIA PaCa-2 cell line. Evaluation of the presence of Survivin-T34A in these exosomes followed by their ability to induce Gemcitabine-potentiative cell killing was the objective of this work. Results: Here we show that exosomes collected in the absence of tetracycline (tet-off from the engineered melanoma cell do contain Survivin-T34A and when used alone or in combination with Gemcitabine, induced a significant increase in apoptotic cell death when compared to Gemcitabine alone on a variety of pancreatic cancer cell lines. Conclusion: This exosomes/Survivin-T34A study shows that a new delivery method for anticancer proteins within the cancer microenvironment may prove useful in targeting cancers of the pancreas.

  10. NK cell-released exosomes

    OpenAIRE

    Fais, Stefano

    2013-01-01

    We have recently reported that human natural killer (NK) cells release exosomes that express both NK-cell markers and cytotoxic molecules. Similar results were obtained with circulating exosomes from human healthy donors. Both NK-cell derived and circulating exosomes exerted a full functional activity and killed both tumor and activated immune cells. These findings indicate that NK-cell derived exosomes might constitute a new promising therapeutic tool.

  11. Characterization of CLL exosomes reveals a distinct microRNA signature and enhanced secretion by activation of BCR signaling.

    Science.gov (United States)

    Yeh, Yuh-Ying; Ozer, Hatice Gulcin; Lehman, Amy M; Maddocks, Kami; Yu, Lianbo; Johnson, Amy J; Byrd, John C

    2015-05-21

    Multiple studies show that chronic lymphocytic leukemia (CLL) cells are heavily dependent on their microenvironment for survival. Communication between CLL cells and the microenvironment is mediated through direct cell contact, soluble factors, and extracellular vesicles. Exosomes are small particles enclosed with lipids, proteins, and small RNAs that can convey biological materials to surrounding cells. Our data herein demonstrate that CLL cells release significant amounts of exosomes in plasma that exhibit abundant CD37, CD9, and CD63 expression. Our work also pinpoints the regulation of B-cell receptor (BCR) signaling in the release of CLL exosomes: BCR activation by α-immunoglobulin (Ig)M induces exosome secretion, whereas BCR inactivation via ibrutinib impedes α-IgM-stimulated exosome release. Moreover, analysis of serial plasma samples collected from CLL patients on an ibrutinib clinical trial revealed that exosome plasma concentration was significantly decreased following ibrutinib therapy. Furthermore, microRNA (miR) profiling of plasma-derived exosomes identified a distinct exosome microRNA signature, including miR-29 family, miR-150, miR-155, and miR-223 that have been associated with CLL disease. Interestingly, expression of exosome miR-150 and miR-155 increases with BCR activation. In all, this study successfully characterized CLL exosomes, demonstrated the control of BCR signaling in the release of CLL exosomes, and uncovered a disease-relevant exosome microRNA profile. © 2015 by The American Society of Hematology.

  12. Exosomal and Non-Exosomal Transport of Extra-Cellular microRNAs in Follicular Fluid: Implications for Bovine Oocyte Developmental Competence.

    Directory of Open Access Journals (Sweden)

    Md Mahmodul Hasan Sohel

    Full Text Available Cell-cell communication within the follicle involves many signaling molecules, and this process may be mediated by secretion and uptake of exosomes that contain several bioactive molecules including extra-cellular miRNAs. Follicular fluid and cells from individual follicles of cattle were grouped based on Brilliant Cresyl Blue (BCB staining of the corresponding oocytes. Both Exoquick precipitation and differential ultracentrifugation were used to separate the exosome and non-exosomal fraction of follicular fluid. Following miRNA isolation from both fractions, the human miRCURY LNA™ Universal RT miRNA PCR array system was used to profile miRNA expression. This analysis found that miRNAs were present in both exosomal and non-exosomal fraction of bovine follicular fluid. We found 25 miRNAs differentially expressed (16 up and 9 down in exosomes and 30 miRNAs differentially expressed (21 up and 9 down in non-exosomal fraction of follicular fluid in comparison of BCB- versus BCB+ oocyte groups. Expression of selected miRNAs was detected in theca, granulosa and cumulus oocyte complex. To further explore the potential roles of these follicular fluid derived extra-cellular miRNAs, the potential target genes were predicted, and functional annotation and pathway analysis revealed most of these pathways are known regulators of follicular development and oocyte growth. In order to validate exosome mediated cell-cell communication within follicular microenvironment, we demonstrated uptake of exosomes and resulting increase of endogenous miRNA level and subsequent alteration of mRNA levels in follicular cells in vitro. This study demonstrates for the first time, the presence of exosome or non-exosome mediated transfer of miRNA in the bovine follicular fluid, and oocyte growth dependent variation in extra-cellular miRNA signatures in the follicular environment.

  13. In Vivo Neuroimaging of Exosomes Using Gold Nanoparticles.

    Science.gov (United States)

    Betzer, Oshra; Perets, Nisim; Angel, Ariel; Motiei, Menachem; Sadan, Tamar; Yadid, Gal; Offen, Daniel; Popovtzer, Rachela

    2017-11-28

    Exosomes are emerging as effective therapeutic tools for various pathologies. These extracellular vesicles can bypass biological barriers, including the blood-brain barrier, and can serve as powerful drug and gene therapy transporters. However, the progress of therapy development is impeded by several challenges, including insufficient data on exosome trafficking and biodistribution and the difficulty to image deep brain structures in vivo. Herein, we established a method for noninvasive in vivo neuroimaging and tracking of exosomes, based on glucose-coated gold nanoparticle (GNP) labeling and computed tomography imaging. Labeling of exosomes with the GNPs was achieved directly, as opposed to the typical and less efficient indirect labeling mode through parent cells. On the mechanistic level, we found that the glucose-coated GNPs were uptaken into MSC-derived exosomes via an active, energy-dependent mechanism that is mediated by the glucose transporter GLUT-1 and involves endocytic proteins. Next, we determined optimal parameters of size and administration route; we demonstrated that 5 nm GNPs enabled improved exosome labeling and that intranasal, compared to intravenous, administration led to superior brain accumulation and thus enhanced in vivo neuroimaging. Furthermore, using a mouse model of focal brain ischemia, we noninvasively tracked intranasally administered GNP-labeled exosomes, which showed increased accumulation at the lesion site over 24 h, as compared to nonspecific migration and clearance from control brains over the same period. Thus, this exosome labeling technique can serve as a powerful diagnostic tool for various brain disorders and could potentially enhance exosome-based treatments for neuronal recovery.

  14. Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting

    NARCIS (Netherlands)

    van Balkom, Bas W M|info:eu-repo/dai/nl/256594783; Eisele, Almut S; Pegtel, D Michiel; Bervoets, Sander; Verhaar, Marianne C|info:eu-repo/dai/nl/182921840

    2015-01-01

    Exosomes are small vesicles that mediate cell-cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a

  15. Chondroitin sulfate proteoglycan serglycin influences protein cargo loading and functions of tumor-derived exosomes.

    Science.gov (United States)

    Purushothaman, Anurag; Bandari, Shyam K; Chandrashekar, Darshan S; Jones, Richard J; Lee, Hans C; Weber, Donna M; Orlowski, Robert Z

    2017-09-26

    Tumor cells produce and utilize exosomes to promote tumor growth and metastasis. Tumor-cell-derived exosomes deliver cargos that partially mimic the contents of the parent cell to nearby or distant normal or abnormal cells, thereby reprogramming the recipient cells to support tumor progression. Mechanisms by which tumor-derived exosomes subserve the tumor are under intense investigation. Here we demonstrate a critical role of the chondroitin sulfate proteoglycan serglycin in regulating the protein cargo and functions of myeloma cell-derived exosomes. Previous studies have shown that serglycin, the only known intracellular proteoglycan, functions mainly in the storage of basically charged components within the intracellular granules/vesicles via serglycin's densely clustered, negatively charged glycosaminoglycan chains. Here we demonstrate that serglycin plays a critical role in the protein cargo loading of tumor-derived exosomes. Serglycin was detected in exosomes derived from cell culture supernatants of human myeloma cell lines and serum of myeloma patients. Mass spectrometry analysis of exosomal proteins identified significantly fewer protein components within exosomes derived from serglycin-knockdown myeloma cells than within exosomes from control cells. On gene ontology analysis, exosomes derived from serglycin-knockdown cells, but not from control cells, lacked many proteins that are required for mediating different cellular processes. In functional assays, exosomes from serglycin-knockdown cells failed to induce an invasive phenotype in myeloma cells and failed to promote migration of macrophages. These findings reveal that serglycin plays an important role in maintaining the protein cargo in tumor-derived exosomes and suggest that targeting serglycin may temper the influence of these exosomes on cancer progression.

  16. Cardiac progenitor-derived exosomes protect ischemic myocardium from acute ischemia/reperfusion injury

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Lijuan [Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing 210009 (China); Cardiovascular Disease, Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267 (United States); Wang, Yingjie [Cardiovascular Disease, Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267 (United States); Internal Medicine of Traditional Chinese Medicine, Shuguang Hospital of Shanghai University of Traditional Chinese Medicine, Shanghai 201203 (China); Pan, Yaohua; Zhang, Lan [Cardiovascular Disease, Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267 (United States); Shen, Chengxing [Department of Cardiology, Xinhua Hospital, Shanghai Jiao Tong University, Shanghai (China); Qin, Gangjian [Feinberg Cardiovascular Research Institute, Northwestern University Feinberg School of Medicine, Chicago, IL 60611 (United States); Ashraf, Muhammad [Pathology and Lab Med, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267 (United States); Weintraub, Neal [Cardiovascular Disease, Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267 (United States); Ma, Genshan, E-mail: magenshan@hotmail.com [Department of Cardiology, Zhongda Hospital, Medical School of Southeast University, Nanjing 210009 (China); Tang, Yaoliang, E-mail: tangyg@ucmail.uc.edu [Cardiovascular Disease, Internal Medicine, University of Cincinnati, 231 Albert Sabin Way, Cincinnati, OH 45267 (United States)

    2013-02-15

    Highlights: ► Cardiac progenitor-derived (CPC) Exosomes protect H9C2 from apoptosis in vitro. ► CPC-exosomes protect cardiomyoyctes from MI/R induced apoptosis in vivo. ► CPC-exosomes were taken up by H9C2 with high efficiency using PKH26 labeling. ► miR-451, one of GATA4-responsive miRNA cluster, is enriched in CPC-exosomes. -- Abstract: Background: Cardiac progenitors (CPC) mediate cardioprotection via paracrine effects. To date, most of studies focused on secreted paracrine proteins. Here we investigated the CPC-derived-exosomes on protecting myocardium from acute ischemia/reperfusion (MI/R) injury. Methods and results: CPC were isolated from mouse heart using two-step protocol. Exosomes were purified from conditional medium, and confirmed by electron micrograph and Western blot using CD63 as a marker. qRT-PCR shows that CPC-exosomes have high level expression of GATA4-responsive-miR-451. Exosomes were ex vivo labeled with PKH26, We observed exosomes can be uptaken by H9C2 cardiomyoblasts with high efficiency after 12 h incubation. CPC-exosomes protect H9C2 from oxidative stress by inhibiting caspase 3/7 activation invitro. In vivo delivery of CPC-exosomes in an acute mouse myocardial ischemia/reperfusion model inhibited cardiomyocyte apoptosis by about 53% in comparison with PBS control (p < 0.05). Conclusion: Our results suggest, for the first time, the CPC-exosomes can be used as a therapeutic vehicle for cardioprotection, and highlights a new perspective for using non-cell exosomes for cardiac disease.

  17. The non-targeted effects of radiation are perpetuated by exosomes

    Energy Technology Data Exchange (ETDEWEB)

    Al-Mayah, Ammar; Bright, Scott; Chapman, Kim [Genomic Instability Group, Oxford Brookes University, Gipsy Lane Campus, Headington, Oxford OX3 0BP (United Kingdom); Irons, Sarah [Insect Virus Research Group, Oxford Brookes University, Gipsy Lane Campus, Headington, Oxford OX3 0BP (United Kingdom); Luo, Ping [Izon Science Ltd., The Oxford Science Park, Magdalen Centre, Robert Robinson Avenue, Oxford OX4 4GA (United Kingdom); Carter, David [Chromatin and non-coding RNA, Oxford Brookes University, Gipsy Lane Campus, Headington, Oxford OX3 0BP (United Kingdom); Goodwin, Edwin [The New Mexico Consortium, Los Alamos, NM 87544 (United States); Kadhim, Munira, E-mail: mkadhim@brookes.ac.uk [Genomic Instability Group, Oxford Brookes University, Gipsy Lane Campus, Headington, Oxford OX3 0BP (United Kingdom)

    2015-02-15

    Highlights: • Radiation induces a DNA damaging process in bystander cells through cell–cell signalling. • Exosome RNA and protein molecules play crucial roles in bystander effects. • Cell progeny inherit the ability to secret bystander effect-inducing exosomes. • This mechanism is most likely accountable for the propagation of GI. - Abstract: Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma. Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood. The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein–RNA inactivation. These were added to separate populations of unirradiated cells. The BE was

  18. Exosomes are fingerprints of originating cells: potential biomarkers for ovarian cancer

    Directory of Open Access Journals (Sweden)

    Kobayashi M

    2015-03-01

    Full Text Available Miharu Kobayashi, Gregory E Rice, Jorge Tapia, Murray D Mitchell, Carlos Salomon Exosome Biology Laboratory, Centre for Clinical Diagnostics, University of Queensland Centre for Clinical Research, Royal Brisbane and Women's Hospital, Brisbane, QLD, Australia. Abstract: The past decade has seen an extraordinary explosion of research in the field of extracellular vesicles, especially in a specific type of extracellular vesicles originating from endosomal compartments, called exosomes. Exosomes are a specific subtype of secreted vesicles that are defined as small (~30–120 nm but very stable membrane vesicles that are released from a wide range of cells, including normal and cancer cells. As the content of exosomes is cell type specific, it is believed that they are a "fingerprint" of the releasing cell and its metabolic status. We hypothesized that the exosomes and their specific exosomal content (eg, microribonucleic acid represent a precious biomedical tool and may be used as biomarkers for the diagnosis and prognosis of malignant tumors. In addition, exosomes may modify the phenotype of the parent and/or target cell by transferring pro-oncogenic molecules to induce cancerous phenotype of recipient cells and contribute to the formation of the premetastatic niche. The mechanism involved in these phenomena remains unclear; however, inclusion of signaling mediators into exosomes or exosome release may reduce their intracellular bioavailability in the parent cell, thereby altering cell phenotype and their metastatic potential. The aim of this review therefore is to analyze the biogenesis and role of exosomes from tumor cells, focusing primarily on ovarian cancer. Ovarian cancer is the most lethal gynecologic cancer, and an effective early diagnosis has the potential to improve patient survival. Ovarian cancer currently lacks a reliable method for early detection, however, exosomes have received great attention as potential biomarkers and mediators

  19. Exosomes in Prostate Cancer: Putting Together the Pieces of a Puzzle

    Energy Technology Data Exchange (ETDEWEB)

    Soekmadji, Carolina, E-mail: carolina.soekmadji@qut.edu.au; Russell, Pamela J.; Nelson, Colleen C. [Australian Prostate Cancer Research Centre-Queensland, Institute of Health and Biomedical Innovation, Queensland University of Technology, Translational Research Institute, Level 3 West, 37 Kent Street, Brisbane, Queensland 4102 (Australia)

    2013-11-11

    Exosomes have been shown to act as mediators for cell to cell communication and as a potential source of biomarkers for many diseases, including prostate cancer. Exosomes are nanosized vesicles secreted by cells and consist of proteins normally found in multivesicular bodies, RNA, DNA and lipids. As a potential source of biomarkers, exosomes have attracted considerable attention, as their protein content resembles that of their cells of origin, even though it is noted that the proteins, miRNAs and lipids found in the exosomes are not a reflective stoichiometric sampling of the contents from the parent cells. While the biogenesis of exosomes in dendritic cells and platelets has been extensively characterized, much less is known about the biogenesis of exosomes in cancer cells. An understanding of the processes involved in prostate cancer will help to further elucidate the role of exosomes and other extracellular vesicles in prostate cancer progression and metastasis. There are few methodologies available for general isolation of exosomes, however validation of those methodologies is necessary to study the role of exosomal-derived biomarkers in various diseases. In this review, we discuss “exosomes” as a member of the family of extracellular vesicles and their potential to provide candidate biomarkers for prostate cancer.

  20. The release and trans-synaptic transmission of Tau via exosomes.

    Science.gov (United States)

    Wang, Yipeng; Balaji, Varun; Kaniyappan, Senthilvelrajan; Krüger, Lars; Irsen, Stephan; Tepper, Katharina; Chandupatla, RamReddy; Maetzler, Walter; Schneider, Anja; Mandelkow, Eckhard; Mandelkow, Eva-Maria

    2017-01-13

    Tau pathology in AD spreads in a hierarchical pattern, whereby it first appears in the entorhinal cortex, then spreads to the hippocampus and later to the surrounding areas. Based on this sequential appearance, AD can be classified into six stages ("Braak stages"). The mechanisms and agents underlying the progression of Tau pathology are a matter of debate. Emerging evidence indicates that the propagation of Tau pathology may be due to the transmission of Tau protein, but the underlying pathways and Tau species are not well understood. In this study we investigated the question of Tau spreading via small extracellular vesicles called exosomes. Exosomes from different sources were analyzed by biochemical methods and electron microscopy (EM) and cryo-EM. Microfluidic devices that allow the culture of cell populations in different compartments were used to investigate the spreading of Tau. We show that Tau protein is released by cultured primary neurons or by N2a cells overexpressing different Tau constructs via exosomes. Neuron-derived exosomal Tau is hypo-phosphorylated, compared with cytosolic Tau. Depolarization of neurons promotes release of Tau-containing exosomes, highlighting the importance of neuronal activity. Using microfluidic devices we show that exosomes mediate trans-neuronal transfer of Tau depending on synaptic connectivity. Tau spreading is achieved by direct transmission of exosomes between neurons. In organotypic hippocampal slices, Tau-containing exosomes in conditioned medium are taken up by neurons and microglia, not astrocytes. In N2a cells, Tau assemblies are released via exosomes. They can induce inclusions of other Tau molecules in N2a cells expressing mutant human Tau. We also studied exosomes from cerebrospinal fluid in AD and control subjects containing monomeric and oligomeric Tau. Split-luciferase complementation reveals that exosomes from CSF can promote Tau aggregation in cultured cells. Our study demonstrates that exosomes contribute

  1. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2.

    Science.gov (United States)

    de Jong, Olivier G; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C

    2016-02-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations, research on exosome-ECM interactions is limited. Here, we investigate whether the exosome-associated lysyl oxidase family member lysyl oxidase-like 2 (LOXL2) is involved in ECM remodelling. We found that LOXL2 is present on the exterior of endothelial cell (EC)-derived exosomes, placing it in direct vicinity of the ECM. It is up-regulated twofold in EC-derived exosomes cultured under hypoxic conditions. Intact exosomes from hypoxic EC and LOXL2 overexpressing EC show increased activity in a fluorometric lysyl oxidase enzymatic activity assay as well as in a collagen gel contraction assay. Concordantly, knockdown of LOXL2 in exosome-producing EC in both normal and hypoxic conditions reduces activity of exosomes in both assays. Our findings show for the first time that ECM crosslinking by EC-derived exosomes is mediated by LOXL2 under the regulation of hypoxia, and implicate a role for exosomes in hypoxia-regulated focal ECM remodelling, a key process in both fibrosis and wound healing. © 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

  2. Natural CD8{sup +}25{sup +} regulatory T cell-secreted exosomes capable of suppressing cytotoxic T lymphocyte-mediated immunity against B16 melanoma

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Yufeng; Zhang, Xueshu; Zhao, Tuo; Li, Wei; Xiang, Jim, E-mail: jim.xiang@saskcancer.ca

    2013-08-16

    Highlights: •CD8{sup +}25{sup +} regulatory T cells secrete tolerogenic exosomes. •CD8{sup +}25{sup +} regulatory T cell-derived exosomes exhibit immunosuppressive effect. •CD8{sup +}25{sup +} regulatory T cell-derived exosomes inhibit antitumor immunity. -- Abstract: Natural CD4{sup +}25{sup +} and CD8{sup +}25{sup +} regulatory T (Tr) cells have been shown to inhibit autoimmune diseases. Immune cells secrete exosomes (EXOs), which are crucial for immune regulation. However, immunomodulatory effect of natural Tr cell-secreted EXOs is unknown. In this study, we purified natural CD8{sup +}25{sup +} Tr cells from C57BL/6 mouse naive CD8{sup +} T cells, and in vitro amplified them with CD3/CD28 beads. EXOs (EXO{sub Tr}) were purified from Tr cell’s culture supernatants by differential ultracentrifugation and analyzed by electron microscopy, Western blot and flow cytometry. Our data showed that EXO{sub Tr} had a “saucer” or round shape with 50–100 nm in diameter, contained EXO-associated markers LAMP-1 and CD9, and expressed natural Tr cell markers CD25 and GITR. To assess immunomodulatory effect, we i.v. immunized C57BL/6 mice with ovalbumin (OVA)-pulsed DCs (DC{sub OVA}) plus Tr cells or EXO{sub Tr}, and then assessed OVA-specific CD8{sup +} T cell responses using PE-H-2K{sup b}/OVA tetramer and FITC-anti-CD8 antibody staining by flow cytometry and antitumor immunity in immunized mice with challenge of OVA-expressing BL6–10{sub OVA} melanoma cells. We demonstrated that DC{sub OVA}-stimulated CD8{sup +} T cell responses and protective antitumor immunity significantly dropped from 2.52% to 1.08% and 1.81% (p < 0.05), and from 8/8 to 2/8 and 5/8 mice DC{sub OVA} (p < 0.05) in immunized mice with co-injection of Tr cells and EXO{sub Tr}, respectively. Our results indicate that natural CD8{sup +}25{sup +} Tr cell-released EXOs, alike CD8{sup +}25{sup +} Tr cells, can inhibit CD8{sup +} T cell responses and antitumor immunity. Therefore, EXOs derived from

  3. Targeting the nuclear RNA exosome

    DEFF Research Database (Denmark)

    Meola, Nicola; Jensen, Torben Heick

    2017-01-01

    Centrally positioned in nuclear RNA metabolism, the exosome deals with virtually all transcript types. This 3'-5' exo- and endo-nucleolytic degradation machine is guided to its RNA targets by adaptor proteins that enable substrate recognition. Recently, the discovery of the 'Poly(A) tail exosome...... targeting (PAXT)' connection as an exosome adaptor to human nuclear polyadenylated transcripts has relighted the interest of poly(A) binding proteins (PABPs) in both RNA productive and destructive processes....

  4. Exosomes as nucleic acid nanocarriers.

    Science.gov (United States)

    van den Boorn, Jasper G; Dassler, Juliane; Coch, Christoph; Schlee, Martin; Hartmann, Gunther

    2013-03-01

    Exosomes are nano-sized vesicles produced naturally by many cell types. They are specifically loaded with nucleic acid cargo, dependent on the exosome-producing cell and its homeostatic state. As natural intercellular shuttles of miRNA, exosomes influence an array of developmental, physiological and pathological processes in the recipient cell or tissue to which they can be selectively targeted by their tetraspanin surface-domains. By a review of current research, we illustrate here why exosomes are ideal nanocarriers for use in the targeted in vivo delivery of nucleic acids. Copyright © 2012 Elsevier B.V. All rights reserved.

  5. Mutant Huntingtin Inhibits αB-Crystallin Expression and Impairs Exosome Secretion from Astrocytes.

    Science.gov (United States)

    Hong, Yan; Zhao, Ting; Li, Xiao-Jiang; Li, Shihua

    2017-09-27

    In the brain, astrocytes secrete diverse substances that regulate neuronal function and viability. Exosomes, which are vesicles produced through the formation of multivesicular bodies and their subsequent fusion with the plasma membrane, are also released from astrocytes via exocytotic secretion. Astrocytic exosomes carry heat shock proteins that can reduce the cellular toxicity of misfolded proteins and prevent neurodegeneration. Although mutant huntingtin (mHtt) affects multiple functions of astrocytes, it remains unknown whether mHtt impairs the production of exosomes from astrocytes. We found that mHtt is not present in astrocytic exosomes, but can decrease exosome secretion from astrocytes in HD140Q knock-in (KI) mice. N-terminal mHtt accumulates in the nuclei and forms aggregates, causing decreased secretion of exosomes from cultured astrocytes. Consistently, there is a significant decrease in secreted exosomes in both female and male HD KI mouse striatum in which abundant nuclear mHtt aggregates are present. Conversely, injection of astrocytic exosomes into the striatum of HD140Q KI mice reduces the density of mHtt aggregates. Further, mHtt in astrocytes decreased the expression of αB-crystallin, a small heat shock protein that is enriched in astrocytes and mediates exosome secretion, by reducing the association of Sp1 with the enhancer of the αB-crystallin gene. Importantly, overexpression of αB-crystallin rescues defective exosome release from HD astrocytes as well as mHtt aggregates in the striatum of HD140Q KI mice. Our results demonstrate that mHtt reduces the expression of αB-crystallin in astrocytes to decrease exosome secretion in the HD brains, contributing to non-cell-autonomous neurotoxicity in HD.SIGNIFICANCE STATEMENT Huntington's disease (HD) is characterized by selective neurodegeneration that preferentially occurs in the striatal medium spiny neurons. Recent studies in different HD mouse models demonstrated that dysfunction of astrocytes

  6. The non-targeted effects of radiation are perpetuated by exosomes.

    Science.gov (United States)

    Al-Mayah, Ammar; Bright, Scott; Chapman, Kim; Irons, Sarah; Luo, Ping; Carter, David; Goodwin, Edwin; Kadhim, Munira

    2015-02-01

    Exosomes contain cargo material from endosomes, cytosol, plasma membrane and microRNA molecules, they are released by a number of non-cancer and cancer cells into both the extracellular microenvironment and body fluids such as blood plasma. Recently we demonstrated radiation-induced non-targeted effects [NTE: genomic instability (GI) and bystander effects (BE)] are partially mediated by exosomes, particularly the RNA content. However the mechanistic role of exosomes in NTE is yet to be fully understood. The present study used MCF7 cells to characterise the longevity of exosome-induced activity in the progeny of irradiated and unirradiated bystander cells. Exosomes extracted from conditioned media of irradiated and bystander progeny were added to unirradiated cells. Analysis was carried out at 1 and 20/24 population doublings following medium/exosome transfer for DNA/chromosomal damage. Results confirmed exosomes play a significant role in mediating NTE of ionising radiation (IR). This effect was remarkably persistent, observed >20 doublings post-irradiation in the progeny of bystander cells. Additionally, cell progeny undergoing a BE were themselves capable of inducing BE in other cells via exosomes they released. Furthermore we investigated the role of exosome cargo. Culture media from cells exposed to 2 Gy X-rays was subjected to ultracentrifugation and four inoculants prepared, (a) supernatants with exosomes removed, and pellets with (b) exosome proteins denatured, (c) RNA degraded, and (d) a combination of protein-RNA inactivation. These were added to separate populations of unirradiated cells. The BE was partially inhibited when either exosome protein or exosome RNA were inactivated separately, whilst combined RNA-protein inhibition significantly reduced or eliminated the BE. These results demonstrate that exosomes are associated with long-lived signalling of the NTE of IR. Both RNA and protein molecules of exosomes work in a synergistic manner to initiate NTE

  7. Signaling Pathways in Exosomes Biogenesis, Secretion and Fate

    Directory of Open Access Journals (Sweden)

    Carla Emiliani

    2013-03-01

    Full Text Available Exosomes are small extracellular vesicles (30–100 nm derived from the endosomal system, which have raised considerable interest in the last decade. Several studies have shown that they mediate cell-to-cell communication in a variety of biological processes. Thus, in addition to cell-to-cell direct interaction or secretion of active molecules, they are now considered another class of signal mediators. Exosomes can be secreted by several cell types and retrieved in many body fluids, such as blood, urine, saliva and cerebrospinal fluid. In addition to proteins and lipids, they also contain nucleic acids, namely mRNA and miRNA. These features have prompted extensive research to exploit them as a source of biomarkers for several pathologies, such as cancer and neurodegenerative disorders. In this context, exosomes also appear attractive as gene delivery vehicles. Furthermore, exosome immunomodulatory and regenerative properties are also encouraging their application for further therapeutic purposes. Nevertheless, several issues remain to be addressed: exosome biogenesis and secretion mechanisms have not been clearly understood, and physiological functions, as well as pathological roles, are far from being satisfactorily elucidated.

  8. Exosome platform for diagnosis and monitoring of traumatic brain injury

    Science.gov (United States)

    Taylor, Douglas D.; Gercel-Taylor, Cicek

    2014-01-01

    We have previously demonstrated the release of membranous structures by cells into their extracellular environment, which are termed exosomes, microvesicles or extracellular vesicles depending on specific characteristics, including size, composition and biogenesis pathway. With activation, injury, stress, transformation or infection, cells express proteins and RNAs associated with the cellular responses to these events. The exosomes released by these cells can exhibit an array of proteins, lipids and nucleic acids linked to these physiologic events. This review focuses on exosomes associated with traumatic brain injury, which may be both diagnostic and a causative factor in the progression of the injury. Based on current data, exosomes play essential roles as conveyers of intercellular communication and mediators of many of the pathological conditions associated with development, progression and therapeutic failures and cellular stress in a variety of pathologic conditions. These extracellular vesicles express components responsible for angiogenesis promotion, stromal remodelling, signal pathway activation through growth factor/receptor transfer, chemoresistance, immunologic activation and genetic exchange. These circulating exosomes not only represent a central mediator of the pro-inflammatory microenvironment linked with secondary brain injury, but their presence in the peripheral circulation may serve as a surrogate for biopsies, enabling real-time diagnosis and monitoring of neurodegenerative progression. PMID:25135964

  9. The RNA Exosome and RNA Exosome-linked Disease.

    Science.gov (United States)

    Morton, Derrick J; Kuiper, Emily G; Jones, Stephanie K; Leung, Sara W; Corbett, Anita H; Fasken, Milo B

    2017-11-01

    The RNA exosome is an evolutionarily conserved, ribonuclease complex that is critical for both processing and degradation of a variety of RNAs. Cofactors that associate with the RNA exosome likely dictate substrate specificity for this complex. Recently, mutations in genes encoding both structural subunits of the RNA exosome and its cofactors have been linked to human disease. Mutations in the RNA exosome genes EXOSC3 and EXOSC8 cause pontocerebellar hypoplasia type 1b (PCH1b) and type 1c (PCH1c), respectively, which are similar autosomal recessive, neurodegenerative diseases. Mutations in the RNA exosome gene EXOSC2 cause a distinct syndrome with various tissue-specific phenotypes including retinitis pigmentosa and mild intellectual disability. Mutations in genes that encode RNA exosome cofactors also cause tissue-specific diseases with complex phenotypes. How mutations in these genes give rise to distinct, tissue-specific diseases is not clear. In this review, we discuss the role of the RNA exosome complex and its cofactors in human disease, consider the amino acid changes that have been implicated in disease, and speculate on the mechanisms by which exosome gene mutations could underlie dysfunction and disease. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  10. Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

    Science.gov (United States)

    Yang, Tianzhi; Martin, Paige; Fogarty, Brittany; Brown, Alison; Schurman, Kayla; Phipps, Roger; Yin, Viravuth P; Lockman, Paul; Bai, Shuhua

    2015-06-01

    The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

  11. Plasma exosomes are enriched in Hsp70 and modulated by stress and cortisol in rainbow trout.

    Science.gov (United States)

    Faught, Erin; Henrickson, Lynsi; Vijayan, Mathilakath M

    2017-02-01

    Exosomes are endosomally derived vesicles that are secreted from cells and contain a suite of molecules, including proteins and nucleic acids. Recent studies suggest the possibility that exosomes in circulation may be affecting recipient target cell function, but the modes of action are unclear. Here, we tested the hypothesis that exosomes are in circulation in fish plasma and that these vesicles are enriched with heat shock protein 70 (Hsp70). Exosomes were isolated from rainbow trout (Oncorhynchus mykiss) plasma using differential centrifugation, and their presence was confirmed by transmission electron microscopy and the exosomal marker acetylcholinesterase. Plasma exosomes were enriched with Hsp70, and this stress protein was transiently elevated in trout plasma in response to a heat shock in vivo Using trout hepatocytes in primary culture, we tested whether stress levels of cortisol, the principle corticosteroid in teleosts, regulates exosomal Hsp70 content. As expected, a 1-h heat shock (+15°C above ambient) increased Hsp70 expression in hepatocytes, and this led to higher Hsp70 enrichment in exosomes over a 24-h period. However, cortisol treatment significantly reduced the expression of Hsp70 in exosomes released from either unstressed or heat-shocked hepatocytes. This cortisol-mediated suppression was not specific to Hsp70 as beta-actin expression was also reduced in exosomes released from hepatocytes treated with the steroid. Our results suggest that circulating Hsp70 is released from target tissues via exosomes, and their release is modulated by stress and cortisol. Overall, we propose a novel role for extracellular vesicular transport of Hsp70 in the organismal stress response. © 2017 Society for Endocrinology.

  12. Tumor exosomes induce tunneling nanotubes in lipid raft-enriched regions of human mesothelioma cells

    Energy Technology Data Exchange (ETDEWEB)

    Thayanithy, Venugopal [Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455 (United States); Babatunde, Victor [Moore Laboratory, Department of Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Dickson, Elizabeth L. [Department of Obstetrics and Gynecology, Division of Gynecologic Oncology, University of Minnesota, Minneapolis, MN 55455 (United States); Wong, Phillip [Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455 (United States); Oh, Sanghoon; Ke, Xu; Barlas, Afsar; Fujisawa, Sho; Romin, Yevgeniy [Molecular Cytology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Moreira, André L. [Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Downey, Robert J. [Department of Surgery, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Steer, Clifford J. [Departments of Medicine and Genetics, Cell Biology and Development, University of Minnesota, Minneapolis, MN 55455 (United States); Subramanian, Subbaya [Department of Surgery, University of Minnesota, Minneapolis, MN 55455 (United States); Manova-Todorova, Katia [Molecular Cytology, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Moore, Malcolm A.S. [Moore Laboratory, Department of Cell Biology, Sloan-Kettering Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10021 (United States); Lou, Emil, E-mail: emil-lou@umn.edu [Department of Medicine, Division of Hematology, Oncology and Transplantation, University of Minnesota, Minneapolis, MN 55455 (United States)

    2014-04-15

    Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24–48 h; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3–1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells. - Highlights: • Exosomes derived from malignant cells can stimulate an increased rate in the formation of tunneling nanotubes. • Tunneling nanotubes can serve as conduits for intercellular transfer of these exosomes. • Most notably, exosomes derived from benign mesothelial cells had no effect on nanotube formation. • Cells forming nanotubes were enriched in lipid rafts at a greater number compared with cells not forming nanotubes. • Our findings suggest causal and potentially synergistic association of exosomes and

  13. The Dual Role of Exosomes in Hepatitis A and C Virus Transmission and Viral Immune Activation.

    Science.gov (United States)

    Longatti, Andrea

    2015-12-17

    Exosomes are small nanovesicles of about 100 nm in diameter that act as intercellular messengers because they can shuttle RNA, proteins and lipids between different cells. Many studies have found that exosomes also play various roles in viral pathogenesis. Hepatitis A virus (HAV; a picornavirus) and Hepatitis C virus (HCV; a flavivirus) two single strand plus-sense RNA viruses, in particular, have been found to use exosomes for viral transmission thus evading antibody-mediated immune responses. Paradoxically, both viral exosomes can also be detected by plasmacytoid dendritic cells (pDCs) leading to innate immune activation and type I interferon production. This article will review recent findings regarding these two viruses and outline how exosomes are involved in their transmission and immune sensing.

  14. Resolving sorting mechanisms into exosomes

    NARCIS (Netherlands)

    Stoorvogel, Willem

    2015-01-01

    The complexity of mechanisms driving protein sorting into exosomes is only beginning to emerge. In a paper recently published in Cell Research, Roucourt et al. report that trimming of heparan sulfate side chains of syndecans by endosomal heparanase facilitates sorting into exosomes by the formation

  15. Mesenchymal Stem Cell-Derived Exosomes Promote Fracture Healing in a Mouse Model.

    Science.gov (United States)

    Furuta, Taisuke; Miyaki, Shigeru; Ishitobi, Hiroyuki; Ogura, Toshihiko; Kato, Yoshio; Kamei, Naosuke; Miyado, Kenji; Higashi, Yukihito; Ochi, Mitsuo

    2016-12-01

    : Paracrine signaling by bone-marrow-derived mesenchymal stem cells (MSCs) plays a major role in tissue repair. Although the production of regulatory cytokines by MSC transplantation is a critical modulator of tissue regeneration, we focused on exosomes, which are extracellular vesicles that contain proteins and nucleic acids, as a novel additional modulator of cell-to-cell communication and tissue regeneration. To address this, we used radiologic imaging, histological examination, and immunohistochemical analysis to evaluate the role of exosomes isolated from MSC-conditioned medium (CM) in the healing process in a femur fracture model of CD9(-/-) mice, a strain that is known to produce reduced levels of exosomes. We found that the bone union rate in CD9(-/-) mice was significantly lower than wild-type mice because of the retardation of callus formation. The retardation of fracture healing in CD9(-/-) mice was rescued by the injection of exosomes, but this was not the case after the injection of exosomes-free conditioned medium (CM-Exo). The levels of the bone repair-related cytokines, monocyte chemotactic protein-1 (MCP-1), MCP-3, and stromal cell-derived factor-1 in exosomes were low compared with levels in CM and CM-Exo, suggesting that bone repair may be in part mediated by other exosome components, such as microRNAs. These results suggest that exosomes in CM facilitate the acceleration of fracture healing, and we conclude that exosomes are a novel factor of MSC paracrine signaling with an important role in the tissue repair process. This work focuses on exosomes, which are extracellular vesicles, as a novel additional modulator of cell-to-cell communication. This study evaluated the role of exosomes isolated from mesenchymal stem cell (MSC)-conditioned medium (MSC-CM) in the fracture-healing process of CD9(-/-) mice, a strain that is known to produce reduced levels of exosomes. Retardation of fracture healing in CD9(-/-) mice was rescued by the injection of

  16. [Exosome: Trojan horse in immunotherapy].

    Science.gov (United States)

    Mou, Dan-Lei; Jia, Zhan-Sheng; Bai, Xue-Fan

    2005-04-01

    Exosomes are small membrane-bound vesicles that are secreted by a multitude of eukaryocytes as a consequence of fusion of multivesicular bodies with the plasma membrane. Exosomes can play critical roles in different physiological processes depending on their origins. Exosomes secreted from professional antigen-presenting cells are enriched in MHC class I and II complexes, costimulatory molecules, hsp 70 and hsp 90 chaperones, therefore exosomes, like Trojan horse, are of importance of immunoregulation in vivo and in vitro. The review will present current trends of research on the fundamental properties, production and purification of exosomes, and will focus on their implementation in cancer and virus immunotherapy as a novel cell-free peptide-based vaccine.

  17. Role of Lymphocyte Subsets in the Immune Response to Primary B Cell-Derived Exosomes.

    Science.gov (United States)

    Saunderson, Sarah C; McLellan, Alexander D

    2017-10-01

    Exosomes are lipid nanovesicles released after fusion of the endosomal limiting membrane with the plasma membrane. In this study, we investigated the requirement for CD4 T cells, B cells, and NK cells to provide help for CD8 T cell-mediated response to B cell-derived exosomes. CTL responses to Ag-loaded exosomes were dependent on host MHC class I, with a critical role for splenic langerin(+) CD8α(+) dendritic cells (DCs) in exosomal Ag cross-presentation. In addition, there was an absolute dependence on the presence of CD4 T cells, CD8 T cells, and NK cells, where the loss of any one of these subsets led to a complete loss of CTL response. Interestingly, NK cell depletion experiments demonstrated a critical cutoff point for depletion efficacy, with low-level residual NK cells providing sufficient help to allow optimal CD8 T cell proliferative responses to exosomal protein. Despite the potential role for B cells in the response to B cell-derived exosomal proteins, B cell depletion did not alter the exosome-induced CTL response. Similarly, a possible role for the BCR or circulating Ab in mediating CTL responses to B cell-derived exosomes was ruled out using DHLMP2A mice, which lack secreted and membrane-bound Ab, yet harbor marginal zone and follicular B cells. In contrast, CTL responses to DC-derived exosomes were significantly inhibited within Ab-deficient DHLMP2A mice compared with wild-type mice. However, this response was not restored upon serum transfer, implicating a role for the BCR, but not circulating Ab, in DC-derived exosome responses. Copyright © 2017 by The American Association of Immunologists, Inc.

  18. Dll4-containing exosomes induce capillary sprout retraction in a 3D microenvironment

    Science.gov (United States)

    Sharghi-Namini, Soheila; Tan, Evan; Ong, Lee-Ling Sharon; Ge, Ruowen; Asada, H. Harry

    2014-01-01

    Delta-like 4 (Dll4), a membrane-bound Notch ligand, plays a fundamental role in vascular development and angiogenesis. Dll4 is highly expressed in capillary endothelial tip cells and is involved in suppressing neighboring stalk cells to become tip cells during angiogenesis. Dll4-Notch signaling is mediated either by direct cell-cell contact or by Dll4-containing exosomes from a distance. However, whether Dll4-containing exosomes influence tip cells of existing capillaries is unknown. Using a 3D microfluidic device and time-lapse confocal microscopy, we show here for the first time that Dll4-containing exosomes causes tip cells to lose their filopodia and trigger capillary sprout retraction in collagen matrix. We demonstrate that Dll4 exosomes can freely travel through 3D collagen matrix and transfer Dll4 protein to distant tip cells. Upon reaching endothelial sprout, it causes filopodia and tip cell retraction. Continuous application of Dll4 exosomes from a distance lead to significant reduction of sprout formation. This effect correlates with Notch signaling activation upon Dll4-containing exosome interaction with recipient endothelial cells. Furthermore, we show that Dll4-containing exosomes increase endothelial cell motility while suppressing their proliferation. These data revealed novel functions of Dll4 in angiogenesis through exosomes. PMID:24504253

  19. Prothrombin Loading of Vascular Smooth Muscle Cell-Derived Exosomes Regulates Coagulation and Calcification.

    Science.gov (United States)

    Kapustin, Alexander N; Schoppet, Michael; Schurgers, Leon J; Reynolds, Joanne L; McNair, Rosamund; Heiss, Alexander; Jahnen-Dechent, Willi; Hackeng, Tilman M; Schlieper, Georg; Harrison, Paul; Shanahan, Catherine M

    2017-03-01

    The drug warfarin blocks carboxylation of vitamin K-dependent proteins and acts as an anticoagulant and an accelerant of vascular calcification. The calcification inhibitor MGP (matrix Gla [carboxyglutamic acid] protein), produced by vascular smooth muscle cells (VSMCs), is a key target of warfarin action in promoting calcification; however, it remains unclear whether proteins in the coagulation cascade also play a role in calcification. Vascular calcification is initiated by exosomes, and proteomic analysis revealed that VSMC exosomes are loaded with Gla-containing coagulation factors: IX and X, PT (prothrombin), and proteins C and S. Tracing of Alexa488-labeled PT showed that exosome loading occurs by direct binding to externalized phosphatidylserine (PS) on the exosomal surface and by endocytosis and recycling via late endosomes/multivesicular bodies. Notably, the PT Gla domain and a synthetic Gla domain peptide inhibited exosome-mediated VSMC calcification by preventing nucleation site formation on the exosomal surface. PT was deposited in the calcified vasculature, and there was a negative correlation between vascular calcification and the levels of circulating PT. In addition, we found that VSMC exosomes induced thrombogenesis in a tissue factor-dependent and PS-dependent manner. Gamma-carboxylated coagulation proteins are potent inhibitors of vascular calcification suggesting warfarin action on these factors also contributes to accelerated calcification in patients receiving this drug. VSMC exosomes link calcification and coagulation acting as novel activators of the extrinsic coagulation pathway and inducers of calcification in the absence of Gla-containing inhibitors. © 2017 American Heart Association, Inc.

  20. Investigation of the roles of exosomes in colorectal cancer liver metastasis.

    Science.gov (United States)

    Wang, Xia; Ding, Xiaoling; Nan, Lijuan; Wang, Yiting; Wang, Jing; Yan, Zhiqiang; Zhang, Wei; Sun, Jihong; Zhu, Wei; Ni, Bing; Dong, Suzhen; Yu, Lei

    2015-05-01

    The leading cause of death among cancer patients is tumor metastasis. Tumor-derived exosomes are emerging as mediators of metastasis. In the present study, we demonstrated that exosomes play a pivotal role in the metastatic progression of colorectal cancer. First, a nude mouse model of colorectal cancer liver metastasis was established and characterized. Then, we demonstrated that exosomes from a highly liver metastatic colorectal cancer cell line (HT-29) could significantly increase the metastatic tumor burden and distribution in the mouse liver of Caco-2 colorectal cancer cells, which ordinarily exhibit poor liver metastatic potential. We further investigated the mechanisms by which HT-29-derived-exosomes influence the liver metastasis of colorectal cancer and found that mice treated with HT-29-derived exosomes had a relatively higher level of CXCR4 in the metastatic microenvironment, indicating that exosomes may promote colorectal cancer metastasis by recruiting CXCR4-expressing stromal cells to develop a permissive metastatic microenvironment. Finally, the migration of Caco-2 cells was significantly increased following treatment with HT-29-derived exosomes in vitro, further supporting a role for exosomes in modulating colorectal tumor-derived liver metastasis. The data from the present study may facilitate further translational medicine research into the prevention and treatment of colorectal cancer liver metastasis.

  1. Morphologic and proteomic characterization of exosomes released by cultured extravillous trophoblast cells

    Energy Technology Data Exchange (ETDEWEB)

    Atay, Safinur [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Gercel-Taylor, Cicek [Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States); Kesimer, Mehmet [Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC (United States); Taylor, Douglas D., E-mail: ddtaylor@louisville.edu [Department of Microbiology and Immunology, University of Louisville School of Medicine, Louisville, KY (United States); Obstetrics, Gynecology and Women' s Health, University of Louisville School of Medicine, Louisville, KY (United States)

    2011-05-01

    Exosomes represent an important intercellular communication vehicle, mediating events essential for the decidual microenvironment. While we have demonstrated exosome induction of pro-inflammatory cytokines, to date, no extensive characterization of trophoblast-derived exosomes has been provided. Our objective was to provide a morphologic and proteomic characterization of these exosomes. Exosomes were isolated from the conditioned media of Swan71 human trophoblast cells by ultrafiltration and ultracentrifugation. These were analyzed for density (sucrose density gradient centrifugation), morphology (electron microscopy), size (dynamic light scattering) and protein composition (Ion Trap mass spectrometry and western immunoblotting). Based on density gradient centrifugation, microvesicles from Sw71 cells exhibit a density between 1.134 and 1.173 g/ml. Electron microscopy demonstrated that microvesicles from Sw71 cells exhibit the characteristic cup-shaped morphology of exosomes. Dynamic light scattering showed a bell-shaped curve, indicating a homogeneous population with a mean size of 165 nm {+-} 0.5 nm. Ion Trap mass spectrometry demonstrated the presence of exosome marker proteins (including CD81, Alix, cytoskeleton related proteins, and Rab family). The MS results were confirmed by western immunoblotting. Based on morphology, density, size and protein composition, we defined the release of exosomes from extravillous trophoblast cells and provide their first extensive characterization. This characterization is essential in furthering our understanding of 'normal' early pregnancy.

  2. Exosomes derived from human macrophages suppress endothelial cell migration by controlling integrin trafficking.

    Science.gov (United States)

    Lee, Hee Doo; Kim, Yeon Hyang; Kim, Doo-Sik

    2014-04-01

    Integrin trafficking, including internalization, recycling, and lysosomal degradation, is crucial for the regulation of cellular functions. Exosomes, nano-sized extracellular vesicles, are believed to play important roles in intercellular communications. This study demonstrates that exosomes released from human macrophages negatively regulate endothelial cell migration through control of integrin trafficking. Macrophage-derived exosomes promote internalization of integrin β1 in primary HUVECs. The internalized integrin β1 persistently accumulates in the perinuclear region and is not recycled back to the plasma membrane. Experimental results indicate that macrophage-derived exosomes stimulate trafficking of internalized integrin β1 to lysosomal compartments with a corresponding decrease in the integrin destined for recycling endosomes, resulting in proteolytic degradation of the integrin. Moreover, ubiquitination of HUVEC integrin β1 is enhanced by the exosomes, and exosome-mediated integrin degradation is blocked by bafilomycin A, a lysosomal degradation inhibitor. Macrophage-derived exosomes were also shown to effectively suppress collagen-induced activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and HUVEC migration, which are both dependent on integrin β1. These observations provide new insight into the functional significance of exosomes in the regulation of integrin trafficking. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  3. Exosomes derived from alcohol-treated hepatocytes horizontally transfer liver specific miRNA-122 and sensitize monocytes to LPS

    Science.gov (United States)

    Momen-Heravi, Fatemeh; Bala, Shashi; Kodys, Karen; Szabo, Gyongyi

    2015-01-01

    Hepatocyte damage and inflammation in monocytes/macrophages are central to the pathogenesis of alcoholic hepatitis (AH). MicroRNAs (miRNAs) regulate all of these processes. MiRNA-122 is abundantly expressed in hepatocytes while monocytes/macrophages have low levels. The role of exosomes in AH and possible cross talk between hepatocyte-derived exosomes and immune cells is not explored yet. Here, we show that the number of exosomes significantly increases in the sera of healthy individuals after alcohol binge drinking and in mice after binge or chronic alcohol consumption. Exosomes isolated from sera after alcohol consumption or from in vitro ethanol-treated hepatocytes contained miRNA-122. Exosomes derived from ethanol-treated Huh7.5 cells were taken up by the recipients THP1 monocytes and horizontally transferred a mature form of liver-specific miRNA-122. In vivo, liver mononuclear cells and Kupffer cells from alcohol-fed mice had increased miRNA-122 levels. In monocytes, miRNA-122 transferred via exosomes inhibited the HO-1 pathway and sensitized to LPS stimulation and increased levels of pro-inflammatory cytokines. Finally, inflammatory effects of exosomes from ethanol-treated hepatocytes were prevented by using RNA interference via exosome-mediated delivery of a miRNA-122 inhibitor. These results demonstrate that first, exosomes mediate communication between hepatocytes and monocytes/macrophages and second, hepatocyte-derived miRNA-122 can reprogram monocytes inducing sensitization to LPS. PMID:25973575

  4. Exosomes: A Novel Strategy for Treatment and Prevention of Diseases

    Directory of Open Access Journals (Sweden)

    Jiaqi Wang

    2017-06-01

    Full Text Available An “exosome” is a nanoscale membrane vesicle derived from cell endocytosis that functions as an important intercellular communication mediator regulating the exchange of proteins and genetic materials between donor and surrounding cells. Exosomes secreted by normal and cancer cells participate in tumor initiation, progression, invasion, and metastasis. Furthermore, immune cells and cancer cells exert a two-way bidirectional regulatory effect on tumor immunity by exchanging exosomes. Current studies on exosomes have further expanded their known functions in physiological and pathological processes. The purpose of this review is to describe their discovery and biological functions in the context of their enormous potential in the clinical diagnosis, prevention, and treatment of cancer as well as bacterial and viral infectious diseases.

  5. The biological significance and clinical applications of exosomes in ovarian cancer.

    Science.gov (United States)

    Dorayappan, Kalpana Deepa Priya; Wallbillich, John J; Cohn, David E; Selvendiran, Karuppaiyah

    2016-07-01

    Exosomes are nano-sized (20-100nm) vesicles released by a variety of cells and are generated within the endosomal system or at the plasma membrane. There is emerging evidence that exosomes play a key role in intercellular communication in ovarian and other cancers. The protein and microRNA content of exosomes has been implicated in various intracellular processes that mediate oncogenesis, tumor spread, and drug resistance. Exosomes may prime distant tissue sites for reception of future metastases and their release can be mediated by the tumor microenvironment (e.g., hypoxia). Ovarian cancer-derived exosomes have unique features that could be leveraged for use as biomarkers to facilitate improved detection and treatment of the disease. Further, exosomes have the potential to serve as targets and/or drug delivery vehicles in the treatment of ovarian cancer. In this review we discuss the biological and clinical significance of exosomes relevant to the progression, detection, and treatment of ovarian cancer. Copyright © 2016 Elsevier Inc. All rights reserved.

  6. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes.

    Science.gov (United States)

    Zhou, Yu; Wang, Xu; Sun, Li; Zhou, Li; Ma, Tong-Cui; Song, Li; Wu, Jian-Guo; Li, Jie-Liang; Ho, Wen-Zhe

    2016-12-01

    Exosomes are a class of cell-released small vesicles that mediate intercellular communication by delivering functional factors to recipient cells. During hepatitis C virus (HCV) infection, the interaction between liver resident macrophages and hepatocytes is a key component in liver innate immunity. In this study, we explored the role of exosomes in the delivery of innate anti-HCV factors to hepatocytes from macrophages. We showed that supernatant from TLR3-activated macrophage cultures could efficiently inhibit HCV replication in Huh7 cells. This macrophage-mediated anti-HCV activity was through exosomes because inhibiting exosomes could abrogate the action of macrophages. Further analyses demonstrated that TLR3-activated macrophages release exosomes that contain anti-HCV microRNA (miRNA)-29 family members. Inhibiting miRNA29 could restore HCV replication. These findings suggest a novel antiviral mechanism in liver innate immunity against HCV infection and provide insights to support further studies on developing exosome-based delivery system for disease treatment.-Zhou, Y., Wang, X., Sun, L., Zhou, L., Ma, T.-C., Song, L., Wu, J.-G., Li, J.-L., Ho, W.-Z. Toll-like receptor 3-activated macrophages confer anti-HCV activity to hepatocytes through exosomes. © FASEB.

  7. Tumor-derived exosomes confer antigen-specific immunosuppression in a murine delayed-type hypersensitivity model.

    Directory of Open Access Journals (Sweden)

    Chenjie Yang

    Full Text Available Exosomes are endosome-derived small membrane vesicles that are secreted by most cell types including tumor cells. Tumor-derived exosomes usually contain tumor antigens and have been used as a source of tumor antigens to stimulate anti-tumor immune responses. However, many reports also suggest that tumor-derived exosomes can facilitate tumor immune evasion through different mechanisms, most of which are antigen-independent. In the present study we used a mouse model of delayed-type hypersensitivity (DTH and demonstrated that local administration of tumor-derived exosomes carrying the model antigen chicken ovalbumin (OVA resulted in the suppression of DTH response in an antigen-specific manner. Analysis of exosome trafficking demonstrated that following local injection, tumor-derived exosomes were internalized by CD11c+ cells and transported to the draining LN. Exosome-mediated DTH suppression is associated with increased mRNA levels of TGF-β1 and IL-4 in the draining LN. The tumor-derived exosomes examined were also found to inhibit DC maturation. Taken together, our results suggest a role for tumor-derived exosomes in inducing tumor antigen-specific immunosuppression, possibly by modulating the function of APCs.

  8. Involvement of exosomes in lung inflammation associated with experimental acute pancreatitis.

    Science.gov (United States)

    Bonjoch, Laia; Casas, Vanessa; Carrascal, Montserrat; Closa, Daniel

    2016-10-01

    A frequent complication of acute pancreatitis is the lung damage associated with the systemic inflammatory response. Although various pro-inflammatory mediators generated at both local and systemic levels have been identified, the pathogenic mechanisms of the disease are still poorly understood. In recent years, exosomes have emerged as a new intercellular communication system able to transfer encapsulated proteins and small RNAs and protect them from degradation. Using an experimental model of taurocholate-induced acute pancreatitis in rats, we aimed to evaluate the role of exosomes in the extent of the systemic inflammatory response. Induction of pancreatitis increased the concentration of circulating exosomes, which showed a different proteomic profile to those obtained from control animals. A series of tracking experiments using PKH26-stained exosomes revealed that circulating exosomes effectively reached the alveolar compartment and were internalized by macrophages. In vitro experiments revealed that exosomes obtained under inflammatory conditions activate and polarize these alveolar macrophages towards a pro-inflammatory phenotype. Interestingly, the proteomic analysis of circulating exosomes during acute pancreatitis suggested a multi-organ origin with a relevant role for the liver as a source of these vesicles. Tracking experiments also revealed that the liver retains the majority of exosomes from the peritoneal cavity. We conclude that exosomes are involved in the lung damage associated with experimental acute pancreatitis and could be relevant mediators in the systemic effects of pancreatitis. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  9. Exosomes, not protein or lipids, in mesenteric lymph activate inflammation: Unlocking the mystery of post-shock multiple organ failure.

    Science.gov (United States)

    Kojima, Mitsuaki; Gimenes-Junior, Joao Antonio; Langness, Simone; Morishita, Koji; Lavoie-Gagne, Ophelie; Eliceiri, Brian; Costantini, Todd W; Coimbra, Raul

    2017-01-01

    Previous studies have shown that mesenteric lymph (ML) has a crucial role in driving the systemic inflammatory response after trauma/hemorrhagic shock (T/HS). The specific mediators in the ML that contribute to its biological activity remain unclear despite decades of study. Exosomes are extracellular vesicles that are shed into body fluids such as serum and urine that can mediate intercellular communication. We hypothesized that exosomes are present in the ML after trauma/shock and are responsible for the biological activity of ML. Male rats underwent cannulation of the vessels and mesenteric lymph duct. T/HS was induced by laparotomy and 60 minutes of HS (mean arterial pressure, 35 mmHg), followed by resuscitation. The ML was collected during three distinct time periods (pre-shock, shock, and resuscitation phase) and subsequently separated into exosome and supernatant fractions. Exosomes were characterized by electron microscope, nanoparticle tracking analysis, and immunoblotting. The biological activity of exosomes and supernatant of ML were characterized using a monocyte NF-κB reporter assay and by measuring macrophage intracellular TNF-α production. Exosomes were identified in ML by size and expression of the exosome markers CD63 and HSP70. The number of exosomes present in the ML was 2-fold increased during shock and 4-fold decreased in resuscitation phase compared to pre-shock. However, biological activity of exosomes isolated during the resuscitation phase was markedly increased and caused an 8-fold increase in monocyte NF-κB activation compared to supernatant. Macrophage TNF-α production was also increased after exposure to exosomes harvested in the resuscitation phase. The ML supernatant fraction had no effect on TNF-α production during any phase. Our findings show that exosomes, and not the liquid fraction of ML, are the major component triggering inflammatory responses in monocytes and macrophages after experimental T/HS.

  10. Exosomal microRNA Signatures in the Diagnosis and Prognosis of Ovarian Cancer

    Science.gov (United States)

    2013-10-01

    demonstrated to be associated with cells of embryonic origin (such as the placenta) and activated lymphoid cells .17-20 Although...exosomes modulate lymphocyte functions by mimicking “activation induced cell death” (AICD).27,28 Lymphoid cells appear to release exosomes following...characterized by the simultaneous collaboration of innate and adaptive cell -mediated and humoral responses. In the adaptive anti-tumor response, T cells

  11. Tumor Exosomes Induce Tunneling Nanotubes in Lipid Raft-Enriched Regions of Human Mesothelioma Cells

    Science.gov (United States)

    Thayanithy, Venugopal; Babatunde, Victor; Dickson, Elizabeth L.; Wong, Philip; Oh, Sanghoon; Ke, Xu; Barlas, Afsar; Fujisawa, Sho; Romin, Yevgeniy; Moreira, André L.; Downey, Robert J.; Steer, Clifford J.; Subramanian, Subbaya; Manova-Todorova, Katia; Moore, Malcolm A.S.; Phil, D.; Lou, Emil

    2014-01-01

    Tunneling nanotubes (TnTs) are long, non-adherent, actin-based cellular extensions that act as conduits for transport of cellular cargo between connected cells. The mechanisms of nanotube formation and the effects of the tumor microenvironment and cellular signals on TnT formation are unknown. In the present study, we explored exosomes as potential mediators of TnT formation in mesothelioma and the potential relationship of lipid rafts to TnT formation. Mesothelioma cells co-cultured with exogenous mesothelioma-derived exosomes formed more TnTs than cells cultured without exosomes within 24-48 hours; and this effect was most prominent in media conditions (low-serum, hyperglycemic medium) that support TnT formation (1.3-1.9-fold difference). Fluorescence and electron microscopy confirmed the purity of isolated exosomes and revealed that they localized predominantly at the base of and within TnTs, in addition to the extracellular environment. Time-lapse microscopic imaging demonstrated uptake of tumor exosomes by TnTs, which facilitated intercellular transfer of these exosomes between connected cells. Mesothelioma cells connected via TnTs were also significantly enriched for lipid rafts at nearly a 2-fold higher number compared with cells not connected by TnTs. Our findings provide supportive evidence of exosomes as potential chemotactic stimuli for TnT formation, and also lipid raft formation as a potential biomarker for TnT-forming cells. PMID:24468420

  12. Exosomes in Pathogen Infections: A Bridge to Deliver Molecules and Link Functions

    Directory of Open Access Journals (Sweden)

    Wenchao Zhang

    2018-02-01

    Full Text Available Exosomes are extracellular vesicles derived from cell endocytosis which act as transmitters between cells. They are composed of proteins, lipids, and RNAs through which they participate in cellular crosstalk. Consequently, they play an important role in health and disease. Our view is that exosomes exert a bidirectional regulatory effect on pathogen infections by delivering their content. First, exosomes containing proteins and RNAs derived from pathogens can promote infections in three ways: (1 mediating further infection by transmitting pathogen-related molecules; (2 participating in the immune escape of pathogens; and (3 inhibiting immune responses by favoring immune cell apoptosis. Second, exosomes play anti-infection roles through: (1 inhibiting pathogen proliferation and infection directly; (2 inducing immune responses such as those related to the function of monocyte-macrophages, NK cells, T cells, and B cells. We believe that exosomes act as “bridges” during pathogen infections through the mechanisms mentioned above. The purpose of this review is to describe present findings regarding exosomes and pathogen infections, and highlight their enormous potential in clinical diagnosis and treatment. We discuss two opposite aspects: infection and anti-infection, and we hypothesize a balance between them. At the same time, we elaborate on the role of exosomes in immune regulation.

  13. Post isolation modification of exosomes for nanomedicine applications

    Science.gov (United States)

    Hood, Joshua L

    2016-01-01

    Exosomes are extracellular nanovesicles. They innately possess ideal structural and biocompatible nanocarrier properties. Exosome components can be engineered at the cellular level. Alternatively, when exosome source cells are unavailable for customized exosome production, exosomes derived from a variety of biological origins can be modified post isolation which is the focus of this article. Modification of exosome surface structures allows for exosome imaging and tracking in vivo. Exosome membranes can be loaded with hydrophobic therapeutics to increase drug stability and efficacy. Hydrophilic therapeutics such as RNA can be encapsulated in exosomes to improve cellular delivery. Despite advances in post isolation exosome modification strategies, many challenges to effectively harnessing their therapeutic potential remain. Future topics of exploration include: matching exosome subtypes with nanomedicine applications, optimizing exosomal nanocarrier formulation and investigating how modified exosomes interface with the immune system. Research into these areas will greatly facilitate personalized exosome-based nanomedicine endeavors. PMID:27348448

  14. Novel Modulators of Asthma and Allergy: Exosomes and MicroRNAs

    Directory of Open Access Journals (Sweden)

    Beatriz Sastre

    2017-07-01

    Full Text Available Intercellular communication is crucial to the immune system response. In the recent years, the discovery of exosomes has changed the way immune response orchestration was understood. Exosomes are able to operate as independent units that act as mediators in both physiological and pathological conditions. These structures contain proteins, lipidic mediators, and nucleic acids and notoriously include microRNAs (miRNAs. miRNAs are short RNA sequences (around 19–22 nucleotides with a high phylogenetic conservation and can partially or totally regulate multiple mRNAs, inhibiting protein synthesis. In respiratory diseases such as asthma and allergic sensitization, exosomes released by several cell types and their specific content perform crucial functions in the development and continuation of the pathogenic mechanisms. Released exosomes and miRNAs inside them have been found in different types of clinical samples, such as bronchoalveolar lavage fluids and sputum supernatants, providing new data about the environmental factors and mediators that participate in the inflammatory responses that lead to the exacerbation of asthma. In this review, we summarize our current knowledge of the role of exosomes and miRNAs in asthma and allergic sensitization, paying attention to the functions that both exosomes and miRNAs are described to perform through the literature. We review the effect of exosomes and miRNAs in cells implicated in asthma pathology and the genes and pathways that they modify in them, depicting how their behavior is altered in disease status. We also describe their possible repercussion in asthma diagnosis through their possible role as biomarkers. Therefore, both exosomes and miRNAs can be viewed as potential tools to be added to the arsenal of therapeutics to treat this disease.

  15. Exosomes from hypoxic endothelial cells have increased collagen crosslinking activity through up-regulation of lysyl oxidase-like 2

    NARCIS (Netherlands)

    de Jong, Olivier G.; van Balkom, Bas W M; Gremmels, Hendrik; Verhaar, Marianne C.

    2016-01-01

    Exosomes are important mediators of intercellular communication. Additionally, they contain a variety of components capable of interacting with the extracellular matrix (ECM), including integrins, matrix metalloproteinases and members of the immunoglobin superfamily. Despite these observations,

  16. Exosomal microRNA communication between tissues during organogenesis

    Science.gov (United States)

    Hayashi, Toru; Hoffman, Matthew P.

    2017-01-01

    ABSTRACT Epithelial-mesenchymal interactions are required to coordinate cell proliferation, patterning, and functional differentiation of multiple cell types in a developing organ. This exquisite coordination is dependent on various secreted molecules that provide developmental signals to mediate these tissue interactions. Recently, it was reported that mature mesenchymal-derived microRNAs (miRNAs) in the fetal mouse salivary gland are loaded into exosomes, and transported to the epithelium where they influence progenitor cell proliferation. The exosomal miRNAs regulated epithelial expression of genes involved in DNA methylation in progenitor cells to influence morphogenesis. Thus, exosomal miRNAs are mobile genetic signals that cross tissue boundaries within an organ. These findings raise many questions about how miRNA signals are initiated to coordinate organogenesis and whether they are master regulators of epithelial-mesenchymal interactions. The development of therapeutic applications using exosomal miRNAs for the regeneration of damaged adult organs is a promising area of research. PMID:28816640

  17. Exosomes and Exosomal miRNA in Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    Shamila D. Alipoor

    2016-01-01

    Full Text Available Exosomes are nanosized vesicles released from every cell in the body including those in the respiratory tract and lungs. They are found in most body fluids and contain a number of different biomolecules including proteins, lipids, and both mRNA and noncoding RNAs. Since they can release their contents, particularly miRNAs, to both neighboring and distal cells, they are considered important in cell-cell communication. Recent evidence has shown their possible importance in the pathogenesis of several pulmonary diseases. The differential expression of exosomes and of exosomal miRNAs in disease has driven their promise as biomarkers of disease enabling noninvasive clinical diagnosis in addition to their use as therapeutic tools. In this review, we summarize recent advances in this area as applicable to pulmonary diseases.

  18. Exosomes in Cancer Development, Metastasis and Drug Resistance: A Comprehensive Review

    Science.gov (United States)

    Azmi, Asfar S.; Bao, Bin; Sarkar, Fazlul H.

    2013-01-01

    Trafficking of biological material across membranes is an evolutionary conserved mechanism and is part of any normal cell homeostasis. Such transport is comprised of active, passive, export through microparticles and vesicular transport (exosomes) that collectively maintain proper compartmentalization of important micro and macromolecules. In pathological states, such as cancer, aberrant activity of export machinery results in expulsion of a number of key proteins and microRNAs resulting in their misexpression. Exosome mediated expulsion of intracellular drugs could be another barrier in the proper action of most of the commonly used therapeutics, targeted agents and their intracellular metabolites. Over the last decade, a number of studies have revealed that exosomes cross-talk and/or influence major tumor related pathways such as hypoxia driven EMT, cancer stemness, angiogenesis and metastasis involving many cell types within the tumor microenvironment. Emerging evidence suggest that exosome secreted proteins can also propel fibroblast growth, resulting in Desmoplastic reaction (DR); a major barrier in effective cancer drug delivery. This comprehensive review highlights the advancements in the understanding of the biology of exosomes secretions and the consequence on cancer drug resistance. We propose that the successful combination of cancer treatments to tackle exosome mediated drug resistance requires an interdisciplinary understanding of these cellular exclusion mechanisms, and how secreted biomolecules are involved in cellular cross-talk within the tumor microenvironment. PMID:23709120

  19. Exosomal long noncoding RNA CRNDE-h as a novel serum-based biomarker for diagnosis and prognosis of colorectal cancer.

    Science.gov (United States)

    Liu, Tong; Zhang, Xin; Gao, Shanyu; Jing, Fangmiao; Yang, Yongmei; Du, Lutao; Zheng, Guixi; Li, Peilong; Li, Chen; Wang, Chuanxin

    2016-12-20

    Cancer-secreted long non-coding RNAs (lncRNAs) are emerging mediators of cancer-host cross talk. The aim of our study was to illustrate the clinical significance of the lncRNA CRNDE-h in exosomes purified from the serum of patients with colorectal cancer (CRC). The study was divided into four parts: (1) The exosome isolated methods and lncRNA detected methods which accurately and reproducibly measure CRC-related exosomal CRNDE-h in serum were optimized in preliminary pilot stage; (2) The stability of exosomal CRNDE-h was evaluated systematically; (3) The origin of exosomal CRNDE-h was explorated in vitro and in vivo; (4) The diagnostic and prognostic value of exosomal CRNDE-h for CRC were validated in 468 patients. In pilot study, our results indicated that exosomal CRNDE-h was detectable and stable in serum of CRC patients, and derived from tumor cells. Then, the increased expression of exosomal CRNDE-h was successfully validated in 148 CRC patients when compared with colorectal benign disease patients and healthy donors. Exosomal CRNDE-h level significantly correlated with CRC regional lymph node metastasis (P = 0.019) and distant metastasis (P = 0.003). Moreover, at the cut-off value of 0.020 exosomal CRNDE-h level of serum, the area under ROC curve distinguishing CRC from colorectal benign disease patients and healthy donors was 0.892, with 70.3% sensitivity and 94.4% specificity, which was superior to carcinoembryogenic antigen. In addition, high exosomal CRNDE-h level has a lower overall survival rates than that for low groups (34.6% vs. 68.2%, P h in exosome shed a light on utilizing exosomal CRNDE-h as a noninvasive serum-based tumor marker for diagnosis and prognosis of CRC.

  20. Role of Exosomes Released by Dendritic Cells and/or by Tumor Targets: Regulation of NK Cell Plasticity.

    Science.gov (United States)

    Reiners, Katrin S; Dassler, Juliane; Coch, Christoph; Pogge von Strandmann, Elke

    2014-01-01

    Exosomes are endosomal-derived nanovesicles released by normal and tumor cells, which transfer functionally active proteins, lipids, and nucleic acids between cells. They are important mediators of intercellular communication and act on the adjacent stroma as well as in the periphery. Recently, exosomes have been recognized to play a pathophysiological role in various diseases such as cancer or infectious diseases. Tumor cell-derived exosomes (Tex) have been shown to act as tumor promotors by educating non-malignant cells to provide a tumor supporting microenvironment, which helps to circumvent immune detection by the host and supports metastasis. However, Tex with anti-tumor, immune-activating properties were also described reflecting the complexity of exosomes. Here, we assess the role of extracellular microvesicles/exosomes as messengers affecting NK cell function in health and disease and discuss the molecular basis for the differential impact of exosomes on NK cell activity. The molecular composition/load of exosomes and the mechanisms regulating their release remain unclear and need to be further analyzed to facilitate the development of new treatment options targeting the exosomal machinery.

  1. Human Pericardial Fluid Contains Exosomes Enriched with Cardiovascular-Expressed MicroRNAs and Promotes Therapeutic Angiogenesis.

    Science.gov (United States)

    Beltrami, Cristina; Besnier, Marie; Shantikumar, Saran; Shearn, Andrew I U; Rajakaruna, Cha; Laftah, Abas; Sessa, Fausto; Spinetti, Gaia; Petretto, Enrico; Angelini, Gianni D; Emanueli, Costanza

    2017-03-01

    The pericardial fluid (PF) is contained in the pericardial sac surrounding the heart. MicroRNA (miRNA) exchange via exosomes (endogenous nanoparticles) contributes to cell-to-cell communication. We investigated the hypotheses that the PF is enriched with miRNAs secreted by the heart and that it mediates vascular responses through exosome exchange of miRNAs. The study was developed using leftover material from aortic valve surgery. We found that in comparison with peripheral plasma, the PF contains exosomes enriched with miRNAs co-expressed in patients' myocardium and vasculature. At a functional level, PF exosomes improved survival, proliferation, and networking of cultured endothelial cells (ECs) and restored the angiogenic capacity of ECs depleted (via Dicer silencing) of their endogenous miRNA content. Moreover, PF exosomes improved post-ischemic blood flow recovery and angiogenesis in mice. Mechanistically, (1) let-7b-5p is proangiogenic and inhibits its target gene, TGFBR1, in ECs; (2) PF exosomes transfer a functional let-7b-5p to ECs, thus reducing their TGFBR1 expression; and (3) let-7b-5p depletion in PF exosomes impairs the angiogenic response to these nanoparticles. Collectively, our data support the concept that PF exosomes orchestrate vascular repair via miRNA transfer. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  2. TWEAK-stimulated macrophages inhibit metastasis of epithelial ovarian cancer via exosomal shuttling of microRNA.

    Science.gov (United States)

    Hu, Yuan; Li, Dong; Wu, Anyue; Qiu, Xingdi; Di, Wen; Huang, Lei; Qiu, Lihua

    2017-05-01

    Exosomal-miRNAs are emerging as mediators of crosstalk between tumor cells and macrophages. In this study, we observed that exosomes derived from TWEAK-stimulated macrophages (TMs) could be internalized by epithelial ovarian cancer (EOC) cells and inhibit cell metastasis. Through a miRNA microarray analysis, we identified 19 miRNAs that are differentially expressed in exosomes derived from macrophages treated with or without TWEAK. The study validated that TWEAK not only increased the levels of microRNA-7 (miR-7) in macrophages and its secreted exosomes but also resulted in an elevated level of miR-7 in recipient EOC cells, which eventually reduced the activity of the EGFR/AKT/ERK1/2 pathway. Pre-transfection of antagomiR-7 in TMs substantially decreased the levels of miR-7 in macrophages, its secreted exosomes and the recipient EOC cells with a concomitant enhancement of EOC metastasis, suggesting an involvement of exosomal miR-7 from TMs in modulating the metastasis of EOC cells. Finally, the exosomes from TMs significantly blocked EOC metastasis in a xenograft mouse model. These findings provide a novel model in which TMs inhibit the metastasis of EOC cells via shuttling of exosomal miR-7 to EOC cells, thereby inhibiting the EGFR/AKT/ERK1/2 pathway. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Phenotypic modulation of auto-reactive cells by insertion of tolerogenic molecules via MSC-derived exosomes.

    Science.gov (United States)

    Mokarizadeh, Aram; Delirezh, Nowruz; Morshedi, Ahhmad; Mosayebi, Ghasem; Farshid, Amir-Abbas; Dalir-Naghadeh, Bahram

    2012-01-01

    Auto-reactive cells-mediated immune responses are responsible for the current tissue damages during autoimmunity. Accordingly, functional modulation of auto-reactive cells has been a pivotal aim in many of recent studies. In the current study, we investigated the possibility for insertion of regulatory molecules onto auto-reactive cells through exosomal nano-shuttles as a novel approach for phenotype modification of auto-reactive cells. The exosomes were isolated from supernatant of mesenchymal stem cells culture. Resultant exosomes co-cultured with lymphocytes were harvested from established EAE mice in the presence of antigenic MOG35-55 peptide. After 24 hr, insertion of exosomal tolerogenic molecules (PD-L1, TGF-β, galectin-1) onto auto-reactive cells were explored through flow cytometry. The potency of exosomal inserted membrane molecules to modulate phenotype of auto-reactive lymphocytes was assessed upon ELISA test for their-derived cytokines IFN-γ and IL-17. Incorporation of exosomal molecules into lymohocytes' membrane was confirmed by flow cytometric analyses for surface levels of mentioned molecules. Additionally, the decreased secretion of IFN-γ and IL-17 were detected in exosome pre-treated lymphocytes upon stimulation with MOG peptide. Mesenchymal stem cells -derived exosomes showed to be efficient organelles for insertion of bioactive tolerogenic molecules onto auto-reactive cells and modulation of their phenotypes.

  4. Mast Cell-Derived Exosomes Promote Th2 Cell Differentiation via OX40L-OX40 Ligation

    Directory of Open Access Journals (Sweden)

    Fei Li

    2016-01-01

    Full Text Available Exosomes are nanovesicles released by different cell types, such as dendritic cells (DCs, mast cells (MCs, and tumor cells. Exosomes of different origin play a role in antigen presentation and modulation of immune response to infectious disease. In this study, we demonstrate that mast cells and CD4+ T cells colocated in peritoneal lymph nodes from BALB/c mouse. Further, bone marrow-derived mast cells (BMMCs constitutively release exosomes, which express CD63 and OX40L. BMMC-exosomes partially promoted the proliferation of CD4+ T cells. BMMC-exosomes significantly enhanced the differentiation of naive CD4+ T cells to Th2 cells in a surface contact method, and this ability was partly inhibited by the addition of anti-OX40L Ab. In conclusion, BMMC-exosomes promoted the proliferation and differentiation of Th2 cells via ligation of OX40L and OX40 between exosomes and T cells. This method represents a novel mechanism, in addition to direct cell surface contacts, soluble mediators, and synapses, to regulate T cell actions by BMMC-exosomes.

  5. Active macropinocytosis induction by stimulation of epidermal growth factor receptor and oncogenic Ras expression potentiates cellular uptake efficacy of exosomes.

    Science.gov (United States)

    Nakase, Ikuhiko; Kobayashi, Nahoko Bailey; Takatani-Nakase, Tomoka; Yoshida, Tetsuhiko

    2015-06-03

    Exosomes are approximately 100-nm vesicles that consist of a lipid bilayer of cellular membranes secreted in large quantities from various types of normal and disease-related cells. Endocytosis has been reported as a major pathway for the cellular uptake of exosomes; however, the detailed mechanisms of their cellular uptake are still unknown. Here, we demonstrate the active induction of macropinocytosis (accompanied by actin reorganisation, ruffling of plasma membrane, and engulfment of large volumes of extracellular fluid) by stimulation of cancer-related receptors and show that the epidermal growth factor (EGF) receptor significantly enhances the cellular uptake of exosomes. We also demonstrate that oncogenic K-Ras-expressing MIA PaCa-2 cells exhibit intensive macropinocytosis that actively transports extracellular exosomes into the cells compared with wild-type K-Ras-expressing BxPC-3 cells. Furthermore, encapsulation of the ribosome-inactivating protein saporin with EGF in exosomes using our simple electroporation method produces superior cytotoxicity via the enhanced cellular uptake of exosomes. Our findings contribute to the biological, pharmaceutical, and medical research fields in terms of understanding the macropinocytosis-mediated cellular uptake of exosomes with applications for exosomal delivery systems.

  6. [Multilateral Strategies Utilizing Exosomes for Cancer Therapy].

    Science.gov (United States)

    Nishida-Aoki, Nao; Ochiya, Takahiro

    2017-05-01

    Exosomes are nano-sized extracellular vesicles which transfer their components such as RNA, DNA, and proteins from one cell to another cell. The components are released to the cytoplasm of the recipient cells, having an effect on the cells. Cancerderived exosomes promote cancer progression, invasion, gain of drug resistance, and metastasis. Recently, according to their characteristics, it is expected to apply exosomes to cancer therapies, such as utilizing exosomes as drug delivery systems(DDS) for anticancer drugs and as cancer vaccines to enhance immunity to cancer cells. More, as the cancer-derived exosomes have cancer-promoting effects on multiple stages, inhibiting the function of the cancer-derived exosomes would be helpful to cancer therapies by suppressing cancer progression. DDS and cancer vaccines utilizing exosomes are now undergoing clinical studies, although DDS is suffering from loading efficiency. Treatments by inhibiting the functions of cancer-derived exosomes have still only few reports at experimental levels. Recently, we showed in a mouse model that disruption of cancer-derived exosomes by antibodies could suppress lung metastasis of the human breast cancer cells. Exosomes will provide us the multiple strategies to fight with cancer, which can be applied to cancers from many organs. It is important to confirm safety and overcome technical problems to bring exosomes in practical use.

  7. SMALL VESICLES, BIG VEHICLES: EXOSOMES.

    Directory of Open Access Journals (Sweden)

    Saiz-Lopez P

    2016-09-01

    Full Text Available Exosomes are small membranous vesicles released by different cell types. Since their discovery, they have evolved from being considered simple vehicles for the liberation of cellular wastes, to become one of the most promising fields in the area of biomedical research, and more specifically in oncology, since the different malignant tumors release exosomes to all biological fluids, being involved in various functions of the neoplastic process. At present, it is possible to study these vesicles by minimally invasive techniques in patients, which approach us to obtain a more detailed diagnosis and prognosis, as well as to the discovery of new antitumoral therapies

  8. Exosomes in colorectal carcinoma formation: ALIX under the magnifying glass.

    Science.gov (United States)

    Valcz, Gábor; Galamb, Orsolya; Krenács, Tibor; Spisák, Sándor; Kalmár, Alexandra; Patai, Árpád V; Wichmann, Barna; Dede, Kristóf; Tulassay, Zsolt; Molnár, Béla

    2016-08-01

    Exosomes are small membrane vesicles that have important roles in transporting a great variety of bioactive molecules between epithelial compartment and their microenvironment during tumor formation including colorectal adenoma-carcinoma sequence. We tested the mRNA expression of the top 25 exosome-related markers based on ExoCharta database in healthy (n=49), adenoma (n=49) and colorectal carcinoma (n=49) patients using Affymetrix HGU133 Plus2.0 microarrays. Most related genes showed significantly elevated expression including PGK1, PKM, ANXA5, ENO1, HSP90AB1 and MSN during adenoma-carcinoma sequence. Surprisingly, the expression of ALIX (ALG 2-interacting protein X), involved in multivesicular body (MVB) and exosome formation, was significantly reduced in normal vs adenoma (P=5.02 × 10(-13)) and in normal vs colorectal carcinoma comparisons (P=1.51 × 10(-10)). ALIX also showed significant reduction (PALIX protein levels were accompanied by their gradual transition from diffuse cytoplasmic expression to granular signals, which fell into the 0.6-2 μm diameter size range of MVBs. These ALIX-positive particles were seen in the tumor nests, including tumor-stroma border, which suggest their exosome function. MVB-like structures were also detected in tumor microenvironment including α-smooth muscle actin-positive stromal cells, budding off cancer cells in the tumor front as well as in cancer cells entrapped within lymphoid vessels. In conclusion, we determined the top aberrantly expressed exosome-associated markers and revealed the transition of diffuse ALIX protein signals into a MVB-like pattern during adenoma-carcinoma sequence. These tumor-associated particles seen both in the carcinoma and the surrounding microenvironment can potentially mediate epithelial-stromal interactions involved in the regulation of tumor growth, metastatic invasion and therapy response.

  9. Exosomes and Exosomal miRNA in Respiratory Diseases

    NARCIS (Netherlands)

    Alipoor, Shamila D.; Mortaz, Esmaeil|info:eu-repo/dai/nl/29141320X; Garssen, Johan|info:eu-repo/dai/nl/086369962; Movassaghi, Masoud; Mirsaeidi, Mehdi; Adcock, Ian M.

    Exosomes are nanosized vesicles released from every cell in the body including those in the respiratory tract and lungs. They are found in most body fluids and contain a number of different biomolecules including proteins, lipids, and both mRNA and noncoding RNAs. Since they can release their

  10. Exosomal non-coding RNAs: a promising cancer biomarker.

    Science.gov (United States)

    Yang, Huan; Fu, Hailong; Xu, Wenrong; Zhang, Xu

    2016-12-01

    Novel and non-invasive biomarkers are urgently needed for early detection of cancer. Exosomes are nano-sized particles released by cells and contain various bioactive molecules including proteins, DNA, mRNAs, and non-coding RNAs. Increasing evidence suggests that exosomes play critical roles in tumorigenesis, tumor growth, metastasis, and therapy resistance. Exosomes could be readily accessible in nearly all the body fluids. The altered production of exosomes and aberrant expression of exosomal contents could reflect the pathological state of the body, indicating that exosomes and exosomal contents can be utilized as novel cancer biomarkers. Herein, we review the basic properties of exosomes, the functional roles of exosomes in cancer, and the methods of detecting exosomes and exosomal contents. In particular, we highlight the clinical values of exosomal non-coding RNAs in cancer diagnosis and prognosis.

  11. Cancer-associated fibroblasts release exosomal microRNAs that dictate an aggressive phenotype in breast cancer

    Science.gov (United States)

    Donnarumma, Elvira; Fiore, Danilo; Nappa, Martina; Roscigno, Giuseppina; Adamo, Assunta; Iaboni, Margherita; Russo, Valentina; Affinito, Alessandra; Puoti, Ilaria; Quintavalle, Cristina; Rienzo, Anna; Piscuoglio, Salvatore; Thomas, Renato; Condorelli, Gerolama

    2017-01-01

    Cancer-associated fibroblasts (CAFs) are the major components of the tumor microenvironment. They may drive tumor progression, although the mechanisms involved are still poorly understood. Exosomes have emerged as important mediators of intercellular communication in cancer. They mediate horizontal transfer of microRNAs (miRs), mRNAs and proteins, thus affecting breast cancer progression. Differential expression profile analysis identified three miRs (miRs -21, -378e, and -143) increased in exosomes from CAFs as compared from normal fibroblasts. Immunofluorescence indicated that exosomes may be transferred from CAFs to breast cancer cells, releasing their cargo miRs. Breast cancer cells (BT549, MDA-MB-231, and T47D lines) exposed to CAF exosomes or transfected with those miRs exhibited a significant increased capacity to form mammospheres, increased stem cell and epithelial-mesenchymal transition (EMT) markers, and anchorage-independent cell growth. These effects were reverted by transfection with anti-miRs. Similarly to CAF exosomes, normal fibroblast exosomes transfected with miRs -21, -378e, and -143 promoted the stemness and EMT phenotype of breast cancer cells. Thus, we provided evidence for the first time of the role of CAF exosomes and their miRs in the induction of the stemness and EMT phenotype in different breast cancer cell lines. Indeed, CAFs strongly promote the development of an aggressive breast cancer cell phenotype. PMID:28121625

  12. Exosomes: Implications in HIV-1 Pathogenesis

    Science.gov (United States)

    Madison, Marisa N.; Okeoma, Chioma M.

    2015-01-01

    Exosomes are membranous nanovesicles of endocytic origin that carry host and pathogen derived genomic, proteomic, and lipid cargos. Exosomes are secreted by most cell types into the extracellular milieu and are subsequently internalized by recipient cells. Upon internalization, exosomes condition recipient cells by donating their cargos and/or activating various signal transduction pathways, consequently regulating physiological and pathophysiological processes. The role of exosomes in viral pathogenesis, especially human immunodeficiency virus type 1 [HIV-1] is beginning to unravel. Recent research reports suggest that exosomes from various sources play important but different roles in the pathogenesis of HIV-1. From these reports, it appears that the source of exosomes is the defining factor for the exosomal effect on HIV-1. In this review, we will describe how HIV-1 infection is modulated by exosomes and in turn how exosomes are targeted by HIV-1 factors. Finally, we will discuss potentially emerging therapeutic options based on exosomal cargos that may have promise in preventing HIV-1 transmission. PMID:26205405

  13. Exosomes from Hepatitis C Infected Patients Transmit HCV Infection and Contain Replication Competent Viral RNA in Complex with Ago2-miR122-HSP90

    Science.gov (United States)

    Kodys, Karen; Bala, Shashi; Szabo, Gyongyi

    2014-01-01

    Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies. PMID:25275643

  14. Exosomes from hepatitis C infected patients transmit HCV infection and contain replication competent viral RNA in complex with Ago2-miR122-HSP90.

    Science.gov (United States)

    Bukong, Terence N; Momen-Heravi, Fatemeh; Kodys, Karen; Bala, Shashi; Szabo, Gyongyi

    2014-10-01

    Antibodies targeting receptor-mediated entry of HCV into hepatocytes confer limited therapeutic benefits. Evidence suggests that exosomes can transfer genetic materials between cells; however, their role in HCV infection remains obscure. Here, we show that exosomes isolated from sera of chronic HCV infected patients or supernatants of J6/JFH1-HCV-infected Huh7.5 cells contained HCV RNA. These exosomes could mediate viral receptor-independent transmission of HCV to hepatocytes. Negative sense HCV RNA, indicative of replication competent viral RNA, was present in exosomes of all HCV infected treatment non-responders and some treatment-naïve individuals. Remarkably, HCV RNA was associated with Ago2, HSP90 and miR-122 in exosomes isolated from HCV-infected individuals or HCV-infected Huh7.5 cell supernatants. Exosome-loading with a miR-122 inhibitor, or inhibition of HSP90, vacuolar H+-ATPases, and proton pumps, significantly suppressed exosome-mediated HCV transmission to naïve cells. Our findings provide mechanistic evidence for HCV transmission by blood-derived exosomes and highlight potential therapeutic strategies.

  15. Exosomes Derived from Mesenchymal Stem Cells Suppress Angiogenesis by Down-Regulating VEGF Expression in Breast Cancer Cells

    Science.gov (United States)

    Lee, Jong-Kuen; Park, Sae-Ra; Jung, Bong-Kwang; Jeon, Yoon-Kyung; Lee, Yeong-Shin; Kim, Min-Kyoung; Kim, Yong-Goo; Jang, Ji-Young; Kim, Chul-Woo

    2013-01-01

    Exosomes are small membrane vesicles released by a variety of cell types. Exosomes contain genetic materials, such as mRNAs and microRNAs (miRNAs), implying that they may play a pivotal role in cell-to-cell communication. Mesenchymal stem cells (MSCs), which potentially differentiate into multiple cell types, can migrate to the tumor sites and have been reported to exert complex effects on tumor progression. To elucidate the role of MSCs within the tumor microenvironment, previous studies have suggested various mechanisms such as immune modulation and secreted factors of MSCs. However, the paracrine effects of MSC-derived exosomes on the tumor microenvironment remain to be explored. The hypothesis of this study was that MSC-derived exosomes might reprogram tumor behavior by transferring their molecular contents. To test this hypothesis, exosomes from MSCs were isolated and characterized. MSC-derived exosomes exhibited different protein and RNA profiles compared with their donor cells and these vesicles could be internalized by breast cancer cells. The results demonstrated that MSC-derived exosomes significantly down-regulated the expression of vascular endothelial growth factor (VEGF) in tumor cells, which lead to inhibition of angiogenesis in vitro and in vivo. Additionally, miR-16, a miRNA known to target VEGF, was enriched in MSC-derived exosomes and it was partially responsible for the anti-angiogenic effect of MSC-derived exosomes. The collective results suggest that MSC-derived exosomes may serve as a significant mediator of cell-to-cell communication within the tumor microenvironment and suppress angiogenesis by transferring anti-angiogenic molecules. PMID:24391924

  16. Functions of Exosomes and Microbial Extracellular Vesicles in Allergy and Contact and Delayed-Type Hypersensitivity.

    Science.gov (United States)

    Nazimek, Katarzyna; Bryniarski, Krzysztof; Askenase, Philip W

    2016-01-01

    Extracellular vesicles, such as exosomes, are newly recognized intercellular conveyors of functional molecular mechanisms. Notably, they transfer RNAs and proteins between different cells that can then participate in the complex pathogenesis of allergic and related hypersensitivity responses and disease mechanisms, as described herein. This review highlights this important new appreciation of the in vivo participation of such extracellular vesicles in the interactions between allergy-mediating cells. We take into account paracrine epigenetic exchanges mediated by surrounding stromal cells and the endocrine receipt of exosomes from distant cells via the circulation. Exosomes are natural ancient nanoparticles of life. They are made by all cells and in some form by all species down to fungi and bacteria, and are present in all fluids. Besides a new focus on their role in the transmission of genetic regulation, exosome transfer of allergens was recently shown to induce allergic inflammation. Importantly, regulatory and tolerogenic exosomes can potently inhibit allergy and hypersensitivity responses, usually acting nonspecifically, but can also proceed in an antigen-specific manner due to the coating of the exosome surface with antibodies. Deep analysis of processes mediated by exosomes should result in the development of early diagnostic biomarkers, as well as allergen-specific, preventive and therapeutic strategies. These will likely significantly diminish the risks of current allergen-specific parenteral desensitization procedures, and of the use of systemic immunosuppressive drugs. Since extracellular vesicles are physiological, they can be fashioned for the specific delivery of therapeutic molecular instructions through easily tolerated, noninvasive routes, such as oral ingestion, nasal administration, and perhaps even inhalation. © 2016 S. Karger AG, Basel.

  17. Functions of Exosomes and Microbial Extracellular Vesicles in Allergy and Contact and Delayed-Type Hypersensitivity

    Science.gov (United States)

    Nazimek, Katarzyna; Bryniarski, Krzysztof; Askenase, Philip W.

    2016-01-01

    Extracellular vesicles, such as exosomes, are newly recognized intercellular conveyors of functional molecular mechanisms. Notably, they transfer RNAs and proteins between cells in general, that then can participate, as described herein, in the complex pathogenesis of allergic and related hypersensitivity responses and disease mechanisms. This review highlights this important new appreciation of the in vivo participation of such extracellular vesicles in the interactions between allergy-mediating cells, taking into account paracrine epigenetic exchanges mediated by surrounding stromal cells and the endocrine receipt of exosomes from distant cells via the circulation. Exosomes are natural ancient nanoparticles of life. They are made by all cells and in some form by all species down to fungi and bacteria, and are present in all fluids. Besides a new focus on their role in the transmission of genetic regulation, exosome transfer of allergens was recently shown to induce allergic inflammation. Importantly, regulatory and tolerogenic exosomes can potently inhibit allergy and hypersensitivity responses, usually acting non-specifically, but also can proceed in an antigen-specific manner due to coating of the exosome surface with antibodies. Deep analysis of processes mediated by exosomes should result in development of early diagnostic biomarkers, as well as allergen-specific, preventive and therapeutic strategies. These likely will significantly diminish the risks of current allergen specific parenteral desensitization procedures, and of the use of systemic immunosuppressive drugs. Since extracellular vesicles are physiological, they can be fashioned for specific delivery of therapeutic molecular instructions through easily tolerated, non-invasive routes, such as oral ingestion, nasal administration, and perhaps even inhalation. PMID:27820941

  18. Human T-lymphotropic virus type 1-infected cells secrete exosomes that contain Tax protein.

    Science.gov (United States)

    Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V; Sampey, Gavin C; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

    2014-08-08

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Human T-lymphotropic Virus Type 1-infected Cells Secrete Exosomes That Contain Tax Protein*

    Science.gov (United States)

    Jaworski, Elizabeth; Narayanan, Aarthi; Van Duyne, Rachel; Shabbeer-Meyering, Shabana; Iordanskiy, Sergey; Saifuddin, Mohammed; Das, Ravi; Afonso, Philippe V.; Sampey, Gavin C.; Chung, Myung; Popratiloff, Anastas; Shrestha, Bindesh; Sehgal, Mohit; Jain, Pooja; Vertes, Akos; Mahieux, Renaud; Kashanchi, Fatah

    2014-01-01

    Human T-lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and HTLV-1-associated myelopathy/tropical spastic paraparesis. The HTLV-1 transactivator protein Tax controls many critical cellular pathways, including host cell DNA damage response mechanisms, cell cycle progression, and apoptosis. Extracellular vesicles called exosomes play critical roles during pathogenic viral infections as delivery vehicles for host and viral components, including proteins, mRNA, and microRNA. We hypothesized that exosomes derived from HTLV-1-infected cells contain unique host and viral proteins that may contribute to HTLV-1-induced pathogenesis. We found exosomes derived from infected cells to contain Tax protein and proinflammatory mediators as well as viral mRNA transcripts, including Tax, HBZ, and Env. Furthermore, we observed that exosomes released from HTLV-1-infected Tax-expressing cells contributed to enhanced survival of exosome-recipient cells when treated with Fas antibody. This survival was cFLIP-dependent, with Tax showing induction of NF-κB in exosome-recipient cells. Finally, IL-2-dependent CTLL-2 cells that received Tax-containing exosomes were protected from apoptosis through activation of AKT. Similar experiments with primary cultures showed protection and survival of peripheral blood mononuclear cells even in the absence of phytohemagglutinin/IL-2. Surviving cells contained more phosphorylated Rb, consistent with the role of Tax in regulation of the cell cycle. Collectively, these results suggest that exosomes may play an important role in extracellular delivery of functional HTLV-1 proteins and mRNA to recipient cells. PMID:24939845

  20. Ultrasensitive microfluidic analysis of circulating exosomes using a nanostructured graphene oxide/polydopamine coating.

    Science.gov (United States)

    Zhang, Peng; He, Mei; Zeng, Yong

    2016-08-02

    Exosomes are cell-derived nano-sized vesicles that have been recently recognized as new mediators for many cellular processes and potential biomarkers for non-invasive disease diagnosis and the monitoring of treatment response. To better elucidate the biology and clinical value of exosomes, there is a pressing need for new analytical technologies capable of the efficient isolation and sensitive analysis of such small and molecularly diverse vesicles. Herein, we developed a microfluidic exosome analysis platform based on a new graphene oxide/polydopamine (GO/PDA) nano-interface. To the best of our best knowledge, we report for the first time, the GO-induced formation of a 3D nanoporous PDA surface coating enabled by the microfluidic layer-by-layer deposition of GO and PDA. It was demonstrated that this nanostructured GO/PDA interface greatly improves the efficiency of exosome immuno-capture, while at the same time effectively suppressing non-specific exosome adsorption. Based on this nano-interface, an ultrasensitive exosome ELISA assay was developed to afford a very low detection limit of 50 μL(-1) with a 4 log dynamic range, which is substantially better than the existing methods. As a proof of concept for clinical applications, we adapted this platform to discriminate ovarian cancer patients from healthy controls by the quantitative detection of exosomes directly from 2 μL plasma without sample processing. Thus, this platform could provide a useful tool to facilitate basic and clinical investigations of exosomes for non-invasive disease diagnosis and to aid precision treatment.

  1. Prospects for exosomes in immunotherapy of cancer

    OpenAIRE

    Mignot, G; Roux, S; Thery, Clotilde; S?gura, Elodie; Zitvogel, L

    2007-01-01

    Exosomes are nanometer sized membrane vesicles invaginating from multivesicular bodies and secreted from epithelial and hematopoietic cells. They were first described ?in vitro? but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition are unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation or ?Trojan horses? for viruses or prions. Exosome secr...

  2. Roles of Macrophage Exosomes in Immune Response to Calcium Oxalate Monohydrate Crystals

    Directory of Open Access Journals (Sweden)

    Nilubon Singhto

    2018-02-01

    Full Text Available In kidney stone disease, macrophages secrete various mediators via classical secretory pathway and cause renal interstitial inflammation. However, whether their extracellular vesicles, particularly exosomes, are involved in kidney stone pathogenesis remained unknown. This study investigated alterations in exosomal proteome of U937-derived macrophages (by phorbol-12-myristate-13-acetate activation after exposure to calcium oxalate monohydrate (COM crystals for 16-h using 2-DE-based proteomics approach. Six significantly altered proteins in COM-treated exosomes were successfully identified by nanoscale liquid chromatography–electrospray ionization–electron transfer dissociation tandem mass spectrometry as proteins involved mainly in immune processes, including T-cell activation and homeostasis, Fcγ receptor-mediated phagocytosis, interferon-γ (IFN-γ regulation, and cell migration/movement. The decreased heat shock protein 90-beta (HSP90β and increased vimentin were confirmed by Western blotting. ELISA showed that the COM-treated macrophages produced greater level of interleukin-1β (IL-1β, one of the markers for inflammasome activation. Functional studies demonstrated that COM-treated exosomes enhanced monocyte and T-cell migration, monocyte activation and macrophage phagocytic activity, but on the other hand, reduced T-cell activation. In addition, COM-treated exosomes enhanced production of proinflammatory cytokine IL-8 by monocytes that could be restored to its basal level by small-interfering RNA targeting on vimentin (si-Vimentin. Moreover, si-Vimentin could also abolish effects of COM-treated exosomes on monocyte and T-cell migration as well as macrophage phagocytic activity. These findings provided some implications to the immune response during kidney stone pathogenesis via exosomal pathway of macrophages after exposure to COM crystals.

  3. Tumour exosome integrins determine organotropic metastasis.

    Science.gov (United States)

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Tesic Mark, Milica; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M; Dumont-Cole, Vanessa D; Kramer, Kimberly; Wexler, Leonard H; Narendran, Aru; Schwartz, Gary K; Healey, John H; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H; Grandgenett, Paul M; Hollingsworth, Michael A; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K; Jarnagin, William R; Brady, Mary S; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J; Bissell, Mina J; Garcia, Benjamin A; Kang, Yibin; Rajasekhar, Vinagolu K; Ghajar, Cyrus M; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-11-19

    Ever since Stephen Paget's 1889 hypothesis, metastatic organotropism has remained one of cancer's greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis.

  4. Tumour exosome integrins determine organotropic metastasis

    Science.gov (United States)

    Hoshino, Ayuko; Costa-Silva, Bruno; Shen, Tang-Long; Rodrigues, Goncalo; Hashimoto, Ayako; Mark, Milica Tesic; Molina, Henrik; Kohsaka, Shinji; Di Giannatale, Angela; Ceder, Sophia; Singh, Swarnima; Williams, Caitlin; Soplop, Nadine; Uryu, Kunihiro; Pharmer, Lindsay; King, Tari; Bojmar, Linda; Davies, Alexander E.; Ararso, Yonathan; Zhang, Tuo; Zhang, Haiying; Hernandez, Jonathan; Weiss, Joshua M.; Dumont-Cole, Vanessa D.; Kramer, Kimberly; Wexler, Leonard H.; Narendran, Aru; Schwartz, Gary K.; Healey, John H.; Sandstrom, Per; Labori, Knut Jørgen; Kure, Elin H.; Grandgenett, Paul M.; Hollingsworth, Michael A.; de Sousa, Maria; Kaur, Sukhwinder; Jain, Maneesh; Mallya, Kavita; Batra, Surinder K.; Jarnagin, William R.; Brady, Mary S.; Fodstad, Oystein; Muller, Volkmar; Pantel, Klaus; Minn, Andy J.; Bissell, Mina J.; Garcia, Benjamin A.; Kang, Yibin; Rajasekhar, Vinagolu K.; Ghajar, Cyrus M.; Matei, Irina; Peinado, Hector; Bromberg, Jacqueline; Lyden, David

    2015-01-01

    Ever since Stephen Paget’s 1889 hypothesis, metastatic organotropism has remained one of cancer’s greatest mysteries. Here we demonstrate that exosomes from mouse and human lung-, liver- and brain-tropic tumour cells fuse preferentially with resident cells at their predicted destination, namely lung fibroblasts and epithelial cells, liver Kupffer cells and brain endothelial cells. We show that tumour-derived exosomes uptaken by organ-specific cells prepare the pre-metastatic niche. Treatment with exosomes from lung-tropic models redirected the metastasis of bone-tropic tumour cells. Exosome proteomics revealed distinct integrin expression patterns, in which the exosomal integrins α6β4 and α6β1 were associated with lung metastasis, while exosomal integrin αvβ5 was linked to liver metastasis. Targeting the integrins α6β4 and αvβ5 decreased exosome uptake, as well as lung and liver metastasis, respectively. We demonstrate that exosome integrin uptake by resident cells activates Src phosphorylation and pro-inflammatory S100 gene expression. Finally, our clinical data indicate that exosomal integrins could be used to predict organ-specific metastasis. PMID:26524530

  5. Exosomes Secreted by Toxoplasma gondii-Infected L6 Cells: Their Effects on Host Cell Proliferation and Cell Cycle Changes.

    Science.gov (United States)

    Kim, Min Jae; Jung, Bong-Kwang; Cho, Jaeeun; Song, Hyemi; Pyo, Kyung-Ho; Lee, Ji Min; Kim, Min-Kyung; Chai, Jong-Yil

    2016-04-01

    Toxoplasma gondii infection induces alteration of the host cell cycle and cell proliferation. These changes are not only seen in directly invaded host cells but also in neighboring cells. We tried to identify whether this alteration can be mediated by exosomes secreted by T. gondii-infected host cells. L6 cells, a rat myoblast cell line, and RH strain of T. gondii were selected for this study. L6 cells were infected with or without T. gondii to isolate exosomes. The cellular growth patterns were identified by cell counting with trypan blue under confocal microscopy, and cell cycle changes were investigated by flow cytometry. L6 cells infected with T. gondii showed decreased proliferation compared to uninfected L6 cells and revealed a tendency to stay at S or G2/M cell phase. The treatment of exosomes isolated from T. gondii-infected cells showed attenuation of cell proliferation and slight enhancement of S phase in L6 cells. The cell cycle alteration was not as obvious as reduction of the cell proliferation by the exosome treatment. These changes were transient and disappeared at 48 hr after the exosome treatment. Microarray analysis and web-based tools indicated that various exosomal miRNAs were crucial for the regulation of target genes related to cell proliferation. Collectively, our study demonstrated that the exosomes originating from T. gondii could change the host cell proliferation and alter the host cell cycle.

  6. Cell-specific uptake of mantle cell lymphoma-derived exosomes by malignant and non-malignant B-lymphocytes

    Science.gov (United States)

    Hazan-Halevy, Inbal; Rosenblum, Daniel; Weinstein, Shiri; Bairey, Osnat; Raanani, Pia; Peer, Dan

    2015-01-01

    Mantle cell lymphoma (MCL) is an aggressive and incurable mature B cell neoplasm. The current treatments are based on chemotherapeutics and new class of drugs (e.g. Ibrutinib®), which in most cases ends with tumor resistance and relapse. Therefore, further development of novel therapeutic modalities are needed. Exosomes are natural extracellular vesicles, which play an important role in intercellular communication. The specificity of exosome uptake by different target cells remains unknown. In this study, we observed that MCL exosomes are taken up rapidly and preferentially by MCL cells. Only minor fraction of exosomes was internalized into T-cell leukemia and bone marrow stroma cell lines, when these cells were co-cultured with MCL cells. Moreover, MCL patients’ exosomes were taken up by both healthy and patients’ B-lymphocytes with no apparent internalization to T lymphocytes and NK cells. Exosome internalization was not inhibited by specific siRNA against caveolin1 and clathrin but was found to be mediated by cholesterol-dependent pathway. These findings demonstrate natural specificity of exosomes to B-lymphocytes and ultimately might be used for therapeutic intervention in B cells malignancies. PMID:25933830

  7. Endothelial cell-derived exosomes protect SH-SY5Y nerve cells against ischemia/reperfusion injury.

    Science.gov (United States)

    Xiao, Bing; Chai, Yi; Lv, Shigang; Ye, Minhua; Wu, Miaojing; Xie, Liyuan; Fan, Yanghua; Zhu, Xingen; Gao, Ziyun

    2017-10-01

    Cerebral ischemia is a leading cause of death and disability. A previous study indicated that remote ischemic postconditioning (RIP) in the treatment of cerebral ischemia reduces ischemia/reperfusion (I/R) injury. However, the underlying mechanism is not well understood. In the present study, the authors hypothesized that the protective effect of RIP on neurological damage is mediated by exosomes that are released by endothelial cells in femoral arteries. To test this, right middle cerebral artery occlusion/reperfusion with RIP was performed in rats. In addition, an I/R injury cell model was tested that included human umbilical vein endothelial cells (HUVECs) and SH-SY5Y cells. Both the in vivo and in vitro models were examined for injury. Markers of exosomes (CD63, HSP70 and TSG101) were assessed by immunohistochemistry, western blot analysis and flow cytometry. Exosomes were extracted from both animal serum and HUVEC culture medium and identified by electron microscopy. They investigated the role of endothelial cell-derived exosomes in the proliferation, apoptosis, cell cycle, migration and invasion of I/R-injured SH-SY5Y cells. In addition, apoptosis-related molecules caspase-3, Bax and Bcl-2 were detected. RIP was determined to increase the number of exosomes and the expression levels of CD63, HSP70 and TSG101 in plasma, but not in brain hippocampal tissue. The size of exosomes released after I/R in HUVECs was similar to the size of exosomes released in rats subjected to RIP. Endothelial cell-derived exosomes partly suppressed the I/R-induced cell cycle arrest and apoptosis, and inhibited cell proliferation, migration and invasion in SH-SY5Y nerve cells. Endothelial cell-derived exosomes directly protect nerve cells against I/R injury, and are responsible for the protective role of RIP in I/R.

  8. Biochemistry and Function of the RNA Exosomes

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Chlebowski, Aleksander; Dziembowski, Andrzej

    2012-01-01

    Discovery of the evolutionary conserved RNA exosome was a milestone in RNA biology. First identified as an activity essential for the processing of ribosomal RNA, the exosome has since proved to be central for RNA processing and degradation in both the nucleus and the cytoplasm of eukaryotic cell...

  9. Exosomes: The Link Between GPCR Activation and Metastatic Potential?

    Directory of Open Access Journals (Sweden)

    Allison Leigh Isola

    2016-04-01

    Full Text Available The activation of G-Protein Coupled Receptors (GPCRs by their respective ligands initiates a cascade of multiple signaling processes within the cell, regulating growth, metabolism and other essential cellular functions. Dysregulation and aberrant expression of these GPCRs and their subsequent signaling cascades are associated with many different types of pathologies, including cancer. The main life threatening complication in patients diagnosed with cancer is the dissemination of cells from the primary tumor to distant vital organs within the body, metastasis. Communication between the primary tumor, immune system, and the site of future metastasis are some of the key events in the early stages of metastasis. It has been postulated that the communication is mediated by nanovesicles that, under non-pathological conditions, are released by normal cells to relay signals to other cells in the body. These nanovesicles are called exosomes, and are utilized by the tumor cell to influence changes within the recipient cell, such as bone marrow progenitor cells, and cells within the site of future metastatic growth, in order to prepare the site for colonization. Tumor cells have been shown to release an increased number of exosomes when compared to their normal cell counterpart. Exosome production and release are regulated by proteins involved in localization, degradation and size of the multivesicular body, whose function may be altered within cancer cells, resulting in the release of an increased number of these vesicles. This review investigates the possibility of GPCR signaling cascades acting as the upstream activator of proteins involved in exosome production and release, linking a commonly targeted trans-membrane protein class with cellular communication utilized by tumor cells in early stages of metastasis.

  10. Exosome engineering for efficient intracellular delivery of soluble proteins using optically reversible protein-protein interaction module.

    Science.gov (United States)

    Yim, Nambin; Ryu, Seung-Wook; Choi, Kyungsun; Lee, Kwang Ryeol; Lee, Seunghee; Choi, Hojun; Kim, Jeongjin; Shaker, Mohammed R; Sun, Woong; Park, Ji-Ho; Kim, Daesoo; Heo, Won Do; Choi, Chulhee

    2016-07-22

    Nanoparticle-mediated delivery of functional macromolecules is a promising method for treating a variety of human diseases. Among nanoparticles, cell-derived exosomes have recently been highlighted as a new therapeutic strategy for the in vivo delivery of nucleotides and chemical drugs. Here we describe a new tool for intracellular delivery of target proteins, named 'exosomes for protein loading via optically reversible protein-protein interactions' (EXPLORs). By integrating a reversible protein-protein interaction module controlled by blue light with the endogenous process of exosome biogenesis, we are able to successfully load cargo proteins into newly generated exosomes. Treatment with protein-loaded EXPLORs is shown to significantly increase intracellular levels of cargo proteins and their function in recipient cells in vitro and in vivo. These results clearly indicate the potential of EXPLORs as a mechanism for the efficient intracellular transfer of protein-based therapeutics into recipient cells and tissues.

  11. TGF-β Suppression of HBV RNA through AID-Dependent Recruitment of an RNA Exosome Complex

    Science.gov (United States)

    Kitamura, Kouichi; Wang, Zhe; Chowdhury, Sajeda; Monjurul, Ahasan Md; Wakae, Kousho; Koura, Miki; Shimadu, Miyuki; Kinoshita, Kazuo; Muramatsu, Masamichi

    2015-01-01

    Transforming growth factor (TGF)-β inhibits hepatitis B virus (HBV) replication although the intracellular effectors involved are not determined. Here, we report that reduction of HBV transcripts by TGF-β is dependent on AID expression, which significantly decreases both HBV transcripts and viral DNA, resulting in inhibition of viral replication. Immunoprecipitation reveals that AID physically associates with viral P protein that binds to specific virus RNA sequence called epsilon. AID also binds to an RNA degradation complex (RNA exosome proteins), indicating that AID, RNA exosome, and P protein form an RNP complex. Suppression of HBV transcripts by TGF-β was abrogated by depletion of either AID or RNA exosome components, suggesting that AID and the RNA exosome involve in TGF-β mediated suppression of HBV RNA. Moreover, AID-mediated HBV reduction does not occur when P protein is disrupted or when viral transcription is inhibited. These results suggest that induced expression of AID by TGF-β causes recruitment of the RNA exosome to viral RNP complex and the RNA exosome degrades HBV RNA in a transcription-coupled manner. PMID:25836330

  12. Prospects for exosomes in immunotherapy of cancer

    Science.gov (United States)

    Mignot, G; Roux, S; Thery, Clotilde; Ségura, Elodie; Zitvogel, L

    2006-01-01

    Exosomes are nanometer sized membrane vesicles invaginating from multivesicular bodies and secreted from epithelial and hematopoietic cells. They were first described “in vitro” but vesicles with the hallmarks of exosomes are present in vivo in germinal centers and biological fluids. Their protein and lipid composition are unique and could account for their expanding functions such as eradication of obsolete proteins, antigen presentation or “Trojan horses” for viruses or prions. Exosome secretion could be a regulated process participating in the transfer of molecules inbetween immune cells. Despite numerous questions pertaining to their biological relevance, the potential of dendritic cell derived-exosomes as cell-free cancer vaccines is currently being assessed. This review will summarize the composition and formation of exosomes, preclinical data, Phase I trials and optimization protocols for improving their immunogenicity in tumor bearing patients. PMID:16796806

  13. Quantitative and qualitative analysis of small RNAs in human endothelial cells and exosomes provides insights into localized RNA processing, degradation and sorting

    Science.gov (United States)

    van Balkom, Bas W. M.; Eisele, Almut S.; Pegtel, D. Michiel; Bervoets, Sander; Verhaar, Marianne C.

    2015-01-01

    Exosomes are small vesicles that mediate cell–cell communication. They contain proteins, lipids and RNA, and evidence is accumulating that these molecules are specifically sorted for release via exosomes. We recently showed that endothelial-cell-produced exosomes promote angiogenesis in vivo in a small RNA-dependent manner. Recent deep sequencing studies in exosomes from lymphocytic origin revealed a broad spectrum of small RNAs. However, selective depletion or incorporation of small RNA species into endothelial exosomes has not been studied extensively. With next generation sequencing, we identified all known non-coding RNA classes, including microRNAs (miRNAs), small nucleolar RNAs, yRNAs, vault RNAs, 5p and 3p fragments of miRNAs and miRNA-like fragments. In addition, we mapped many fragments of messenger RNAs (mRNAs) and mitochondrial RNAs (mtRNAs). The distribution of small RNAs in exosomes revealed a considerable overlap with the distribution in the producing cells. However, we identified a remarkable enrichment of yRNA fragments and mRNA degradation products in exosomes consistent with yRNAs having a role in degradation of structured and misfolded RNAs in close proximity to endosomes. We propose that endothelial endosomes selectively sequester cytoplasmic RNA-degrading machineries taking part in gene regulation. The release of these regulatory RNAs via exosomes may have implications for endothelial cell–cell communication. PMID:26027894

  14. Structure and reconstitution of yeast Mpp6-nuclear exosome complexes reveals that Mpp6 stimulates RNA decay and recruits the Mtr4 helicase

    Energy Technology Data Exchange (ETDEWEB)

    Wasmuth, Elizabeth V. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Zinder, John C. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Tri-Institutional Training Program in Chemical Biology, Memorial Sloan Kettering Cancer Center, New York, United States; Zattas, Dimitrios [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Das, Mom [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Lima, Christopher D. [Structural Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States; Howard Hughes Medical Institute, Memorial Sloan Kettering Cancer Center, New York, United States

    2017-07-25

    Nuclear RNA exosomes catalyze a range of RNA processing and decay activities that are coordinated in part by cofactors, including Mpp6, Rrp47, and the Mtr4 RNA helicase. Mpp6 interacts with the nine-subunit exosome core, while Rrp47 stabilizes the exoribonuclease Rrp6 and recruits Mtr4, but it is less clear if these cofactors work together. Using biochemistry with Saccharomyces cerevisiae proteins, we show that Rrp47 and Mpp6 stimulate exosome-mediated RNA decay, albeit with unique dependencies on elements within the nuclear exosome. Mpp6-exosomes can recruit Mtr4, while Mpp6 and Rrp47 each contribute to Mtr4-dependent RNA decay, with maximal Mtr4-dependent decay observed with both cofactors. The 3.3 Å structure of a twelve-subunit nuclear Mpp6 exosome bound to RNA shows the central region of Mpp6 bound to the exosome core, positioning its Mtr4 recruitment domain next to Rrp6 and the exosome central channel. Genetic analysis reveals interactions that are largely consistent with our model.

  15. Exosomes expressing carbonic anhydrase 9 promote angiogenesis.

    Science.gov (United States)

    Horie, Kengo; Kawakami, Kyojiro; Fujita, Yasunori; Sugaya, Maki; Kameyama, Koji; Mizutani, Kosuke; Deguchi, Takashi; Ito, Masafumi

    2017-10-21

    Exosomes or microvesicles that are secreted from cells are considered to play important roles in tumor microenvironment. Carbonic anhydrase 9 (CA9), which is induced by hypoxia-inducible factor 1 (HIF1) in response to hypoxia, is overexpressed in many types of cancer including renal cell carcinoma (RCC). We examined the expression level of CA9 in several RCC cell lines and found that the basal level of CA9 was much higher in OSRC-2 cells than in Caki-1, KMRC-1 and 786-O cells. Consistent with the intracellular expression levels, CA9 was abundantly detected in exosomes isolated by ultracentrifugation from OSRC-2 cells. Density gradient centrifugation of OSRC-2 and 786-O exosomes confirmed the co-presence of CA9 with exosomal markers. Upon hypoxia and treatment with CoCl2, a hypoxia mimic agent, the CA9 level in exosomes was increased for all cell lines. In order to examine the effects of CA9 exosomes on angiogenesis, we generated stably transfected HEK293 cells expressing CA9. Immunocytochemical staining demonstrated the uptake of CA9 exosomes by human umbilical vein endothelial cells (HUVEC). In vitro angiogenesis assays using HUVEC revealed that CA9 exosomes promoted migration and tube formation. Lastly, MMP2 expression was increased by treatment with CA9 exosomes in HUVEC. Taken together, our results suggest the possibility that CA9 exosomes released from hypoxic RCC may enhance angiogenesis in microenvironment, thereby contributing to cancer progression. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Transcriptome analysis of exosome-compromised human cells using high-density tiling arrays

    DEFF Research Database (Denmark)

    Jensen, Torben Heick

    The extent of RNA degradation in the nucleus has traditionally been underestimated. However, all major RNA species are synthesized, processed and can be degraded in this compartment and consequently an enormous amount of nucleosides are turned over and recycled. The RNA exosome, a multisubunit......-mediated knock down. We have initiated a transcriptome analysis to investigate the global influence of the nuclear exosome on the abundance  of various RNAs (e.g. normal mRNAs, intergenic transcripts, miRNAs, endogenous retroviral RNAs and small non-coding RNAs). We use an ENCODE (ENCyclopedia of DNA Elements...

  17. Altered microRNA profiles in cerebrospinal fluid exosome in Parkinson disease and Alzheimer disease.

    Science.gov (United States)

    Gui, YaXing; Liu, Hai; Zhang, LiShan; Lv, Wen; Hu, XingYue

    2015-11-10

    The differential diagnosis of Parkinson's diseases (PD) is challenging, especially in the early stages of the disease. We developed a microRNA profiling strategy for exosomal miRNAs isolated from cerebrospinal fluid (CSF) in PD and AD. Sixteen exosomal miRNAs were up regulated and 11 miRNAs were under regulated significantly in PD CSF when compared with those in healthy controls (relative fold > 2, p < 0.05). MiR-1 and miR-19b-3p were validated and significantly reduced in independent samples. While miR-153, miR-409-3p, miR-10a-5p, and let-7g-3p were significantly over expressed in PD CSF exosome. Bioinformatic analysis by DIANA-mirPath demonstrated that Neurotrophin signaling, mTOR signaling, Ubiquitin mediated proteolysis, Dopaminergic synapse, and Glutamatergic synapse were the most prominent pathways enriched in quantiles with PD miRNA patterns. Messenger RNA (mRNA) transcripts [amyloid precursor protein (APP), α-synuclein (α-syn), Tau, neurofilament light gene (NF-L), DJ-1/PARK7, Fractalkine and Neurosin] and long non-coding RNAs (RP11-462G22.1 and PCA3) were differentially expressed in CSF exosomes in PD and AD patients. These data demonstrated that CSF exosomal RNA molecules are reliable biomarkers with fair robustness in regard to specificity and sensitivity in differentiating PD from healthy and diseased (AD) controls.

  18. Ultrastructural Evidence of Exosome Secretion by Progenitor Cells in Adult Mouse Myocardium and Adult Human Cardiospheres

    Directory of Open Access Journals (Sweden)

    Lucio Barile

    2012-01-01

    Full Text Available The demonstration of beneficial effects of cell therapy despite the persistence of only few transplanted cells in vivo suggests secreted factors may be the active component of this treatment. This so-called paracrine hypothesis is supported by observations that culture media conditioned by progenitor cells contain growth factors that mediate proangiogenic and cytoprotective effects. Cardiac progenitor cells in semi-suspension culture form spherical clusters (cardiospheres that deliver paracrine signals to neighboring cells. A key component of paracrine secretion is exosomes, membrane vesicles that are stored intracellularly in endosomal compartments and are secreted when these structures fuse with the cell plasma membrane. Exosomes have been identified as the active component of proangiogenic effects of bone marrow CD34+ stem cells in mice and the regenerative effects of embryonic mesenchymal stem cells in infarcted hearts in pigs and mice. Here, we provide electron microscopic evidence of exosome secretion by progenitor cells in mouse myocardium and human cardiospheres. Exosomes are emerging as an attractive vector of paracrine signals delivered by progenitor cells. They can be stored as an “off-the-shelf” product. As such, exosomes have the potential for circumventing many of the limitations of viable cells for therapeutic applications in regenerative medicine.

  19. Comparative Gene Expression Analysis of Lymphocytes Treated with Exosomes Derived from Ovarian Cancer and Ovarian Cysts

    Directory of Open Access Journals (Sweden)

    Yujuan Li

    2017-06-01

    Full Text Available Cancer cells employ many strategies to evade immune defense and to facilitate tumor growth and angiogenesis. As a novel mode of intercellular communication, cancer-derived exosomes contribute to the recruitment and mediation of lymphocytes within the tumor environment. However, the mechanisms and key molecules mediating the effect of exosomes on lymphocytes are unclear. We treated healthy peripheral blood lymphocytes with exosomes from ovarian cancer and ovarian cysts and screened for differentially expressed genes using the RT2 Profiler Cancer Inflammation and Immunity Crosstalk PCR Array. A total of 26 upregulated genes (mainly pro-inflammatory genes and immunostimulatory and immunosuppressive factor and two downregulated genes (antigen presentation HLA-A/B were identified. Western blotting using lymphocytes from malignant ascites and peritoneal washings of benign ovarian cysts suggested that the interferon and NF-κB signaling pathway were involved in the immune regulation of malignant exosomes. Out of 28 differentially expressed genes detected using the array, 11 were validated by real-time PCR using lymphocytes within ovarian cancer (n = 27 and ovarian cyst (n = 9 environments. In conclusion, our findings indicate that malignant cells secrete exosomes in the tumor microenvironment to recruit lymphocytes in order to suppress antitumor immunity (IL10, Foxp3, and HLA-A/B and enhance tumor invasion, angiogenesis, and dissemination of proinflammatory cytokines (such as IL6 and VEGFA via the interferon and NF-κB signaling pathways. These results clarify lymphocyte-cancer cell cross talk via exosomes and may facilitate the development of effective immunotherapeutic strategies for ovarian cancer.

  20. Exosomes: the ideal nanovectors for biodelivery.

    Science.gov (United States)

    Fais, Stefano; Logozzi, Mariantonia; Lugini, Luana; Federici, Cristina; Azzarito, Tommaso; Zarovni, Natasa; Chiesi, Antonio

    2013-01-01

    Nanomedicine aims to exploit the improved and often novel physical, chemical, and biological properties of materials at the nanometric scale, possibly with the highest level of biomimetism, an approach that simulates what occurs in nature. Although extracellularly released vesicles include both microvesicles (MVs) and exosomes, only exosomes have the size that may be considered suitable for potential use in nanomedicine. In fact, recent reports have shown that exosomes are able to interact with target cells within an organ or at a distance using different mechanisms. Much is yet to be understood about exosomes, and currently, we are looking at the visible top of an iceberg, with most of what we have to understand on these nanovesicles still under the sea. In fact, we know that exosomes released by normal cells always trigger positive effects, whereas those released by cells in pathological condition, such as tumor or infected cells, may induce undesired, dangerous, and mostly unknown effects, but we cannot exclude the possibility that exosomes may also be detrimental for the body in normal conditions. However, whether we consider extracellular vesicles as a whole, thus including MVs, it appears that even in normal conditions, extracellular vesicles may lead to unwanted effects, depending on gender and age. This review aims to critically emphasize existing data in the literature that support the possible roles of exosomes in both diagnostic and therapeutic scopes.

  1. Placental Exosomes as Early Biomarker of Preeclampsia: Potential Role of Exosomal MicroRNAs Across Gestation.

    Science.gov (United States)

    Salomon, Carlos; Guanzon, Dominic; Scholz-Romero, Katherin; Longo, Sherri; Correa, Paula; Illanes, Sebastian E; Rice, Gregory E

    2017-09-01

    There is a need to develop strategies for early prediction of patients who will develop preeclampsia (PE) to establish preventive strategies to reduce the prevalence and severity of the disease and their associated complications. The objective of this study was to investigate whether exosomes and their microRNA cargo present in maternal circulation can be used as early biomarker for PE. A retrospective stratified study design was used to quantify total exosomes and placenta-derived exosomes present in maternal plasma of normal (n = 32 per time point) and PE (n = 15 per time point) pregnancies. Exosomes present in maternal circulation were determined by nanoparticle tracking analysis. An Illumina TruSeq® Small RNA Library Prep Kit was used to construct a small RNA library from exosomal RNA obtained from plasma samples. In presymptomatic women, who subsequently developed PE, the concentration of total exosomes and placenta-derived exosomes in maternal plasma was significantly greater than those observed in controls, throughout pregnancy. The area under the receiver operating characteristic curves for total exosome and placenta-derived exosome concentrations were 0.745 ± 0.094 and 0.829 ± 0.077, respectively. In total, over 300 microRNAs were identified in exosomes across gestation, where hsa-miR-486-1-5p and hsa-miR-486-2-5p were identified as the candidate microRNAs. Although the role of exosomes during PE remains to be fully elucidated, we suggest that the concentration and content of exosomes may be of diagnostic utility for women at risk for developing PE.

  2. Placental exosomes during gestation: liquid biopsies carrying signals for the regulation of human parturition.

    Science.gov (United States)

    Salomon, Carlos; Nuzhat, Zarin; Dixon, Christopher L; Menon, Ramkumar

    2018-01-25

    Parturition is defined as the action or process of giving birth to offspring. Normal term human parturition ensues following the maturation of fetal organ systems typically between 37 and 40 weeks of gestation. Our conventional understanding of how parturition initiation is signaled revolves around feto-maternal immune and endocrine changes occurring in the intrauterine cavity. These changes in turn correlate with the sequence of fetal growth and development. These important physiological changes also result in homeostatic imbalances which result in heightened inflammatory signaling. This disrupts the maintenance of pregnancy, thus leading to labor-related changes. However, the precise mechanisms of the signaling cascades that lead to the initiation of parturition remain unclear, although exosomes may be a mediator of this process. Exosomes are a subtype of extracellular vesicles characterised by their endocytic origin. This involves the trafficking of intraluminal vesicles into multivesicular bodies (MVB) and then exocytosis via the plasmatic membranes. Exosomes are highly stable nanovesicles that are released by a wide range of cells and organs including the human placenta and fetal membranes. Interestingly, exosomes from placental origin have been uncovered in maternal circulation across gestation. In addition, their concentration is higher in pregnancies with complications such as gestational diabetes and preeclampsia. In normal gestation, the concentration of placental exosomes in maternal circulation correlates with placental weight at third trimester. The role of placental exosomes across gestation has not been fully elucidated, although recent studies suggest that placental exosomes are involved in maternal-fetal inmmuno-tolerance, maternal systemic inflammation and nutrient transport. The content of exosomes is of particular importance, encompassing a large range of molecules such as mRNA, miRNAs, DNA, lipids, cell-surface receptors, and protein mediators

  3. Serum exosomal protein profiling for the non-invasive detection of cardiac allograft rejection.

    Science.gov (United States)

    Kennel, Peter J; Saha, Amit; Maldonado, Dawn A; Givens, Raymond; Brunjes, Danielle L; Castillero, Estibaliz; Zhang, Xiaokan; Ji, Ruiping; Yahi, Alexandre; George, Isaac; Mancini, Donna M; Koller, Antonius; Fine, Barry; Zorn, Emmanuel; Colombo, Paolo C; Tatonetti, Nicholas; Chen, Emily I; Schulze, P Christian

    2017-07-19

    Exosomes are cell-derived circulating vesicles that play an important role in cell-cell communication. Exosomes are actively assembled and carry messenger RNAs, microRNAs and proteins. The "gold standard" for cardiac allograft surveillance is endomyocardial biopsy (EMB), an invasive technique with a distinct complication profile. The development of novel, non-invasive methods for the early diagnosis of allograft rejection is warranted. We hypothesized that the exosomal proteome is altered in acute rejection, allowing for a distinction between non-rejection and rejection episodes. Serum samples were collected from heart transplant (HTx) recipients with no rejection, acute cellular rejection (ACR) and antibody-mediated rejection (AMR). Liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis of serum exosome was performed using a mass spectrometer (Orbitrap Fusion Tribrid). Principal component analysis (PCA) revealed a clustering of 3 groups: (1) control and heart failure (HF); (2) HTx without rejection; and (3) ACR and AMR. A total of 45 proteins were identified that could distinguish between groups (q < 0.05). Comparison of serum exosomal proteins from control, HF and non-rejection HTx revealed 17 differentially expressed proteins in at least 1 group (q < 0.05). Finally, comparisons of non-rejection HTx, ACR and AMR serum exosomes revealed 15 differentially expressed proteins in at least 1 group (q < 0.05). Of these 15 proteins, 8 proteins are known to play a role in the immune response. Of note, the majority of proteins identified were associated with complement activation, adaptive immunity such as immunoglobulin components and coagulation. Characterizing of circulating exosomal proteome in different cardiac disease states reveals unique protein expression patterns indicative of the respective pathologies. Our data suggest that HTx and allograft rejection alter the circulating exosomal protein content. Exosomal protein analysis could be a novel approach

  4. Autologous intramuscular transplantation of engineered satellite cells induces exosome-mediated systemic expression of Fukutin-related protein and rescues disease phenotype in a murine model of limb-girdle muscular dystrophy type 2I.

    Science.gov (United States)

    Frattini, Paola; Villa, Chiara; De Santis, Francesca; Meregalli, Mirella; Belicchi, Marzia; Erratico, Silvia; Bella, Pamela; Raimondi, Manuela Teresa; Lu, Qilong; Torrente, Yvan

    2017-10-01

    α-Dystroglycanopathies are a group of muscular dystrophies characterized by α-DG hypoglycosylation and reduced extracellular ligand-binding affinity. Among other genes involved in the α-DG glycosylation process, fukutin related protein (FKRP) gene mutations generate a wide range of pathologies from mild limb girdle muscular dystrophy 2I (LGMD2I), severe congenital muscular dystrophy 1C (MDC1C), to Walker-Warburg Syndrome and Muscle-Eye-Brain disease. FKRP gene encodes for a glycosyltransferase that in vivo transfers a ribitol phosphate group from a CDP -ribitol present in muscles to α-DG, while in vitro it can be secreted as monomer of 60kDa. Consistently, new evidences reported glycosyltransferases in the blood, freely circulating or wrapped within vesicles. Although the physiological function of blood stream glycosyltransferases remains unclear, they are likely released from blood borne or distant cells. Thus, we hypothesized that freely or wrapped FKRP might circulate as an extracellular glycosyltransferase, able to exert a "glycan remodelling" process, even at distal compartments. Interestingly, we firstly demonstrated a successful transduction of MDC1C blood-derived CD133+ cells and FKRP L276IKI mouse derived satellite cells by a lentiviral vector expressing the wild-type of human FKRP gene. Moreover, we showed that LV-FKRP cells were driven to release exosomes carrying FKRP. Similarly, we observed the presence of FKRP positive exosomes in the plasma of FKRP L276IKI mice intramuscularly injected with engineered satellite cells. The distribution of FKRP protein boosted by exosomes determined its restoration within muscle tissues, an overall recovery of α-DG glycosylation and improved muscle strength, suggesting a systemic supply of FKRP protein acting as glycosyltransferase. © The Author 2017. Published by Oxford University Press.

  5. Protein content and functional characteristics of serum-purified exosomes from patients with colorectal cancer revealed by quantitative proteomics.

    Science.gov (United States)

    Chen, Yanyu; Xie, Yong; Xu, Lai; Zhan, Shaohua; Xiao, Yi; Gao, Yanpan; Wu, Bin; Ge, Wei

    2017-02-15

    Tumor cells of colorectal cancer (CRC) release exosomes into the circulation. These exosomes can mediate communication between cells and affect various tumor-related processes in their target cells. We present a quantitative proteomics analysis of the exosomes purified from serum of patients with CRC and normal volunteers; data are available via ProteomeXchange with identifier PXD003875. We identified 918 proteins with an overlap of 725 Gene IDs in the Exocarta proteins list. Compared with the serum-purified exosomes (SPEs) of normal volunteers, we found 36 proteins upregulated and 22 proteins downregulated in the SPEs of CRC patients. Bioinformatics analysis revealed that upregulated proteins are involved in processes that modulate the pretumorigenic microenvironment for metastasis. In contrast, differentially expressed proteins (DEPs) that play critical roles in tumor growth and cell survival were principally downregulated. Our study demonstrates that SPEs of CRC patients play a pivotal role in promoting the tumor invasiveness, but have minimal influence on putative alterations in tumor survival or proliferation. According to bioinformatics analysis, we speculate that the protein contents of exosomes might be associated with whether they are involved in premetastatic niche establishment or growth and survival of metastatic tumor cells. This information will be helpful in elucidating the pathophysiological functions of tumor-derived exosomes, and aid in the development of CRC diagnostics and therapeutics. © 2016 UICC.

  6. Human Endometrial Exosomes Contain Hormone-Specific Cargo Modulating Trophoblast Adhesive Capacity: Insights into Endometrial-Embryo Interactions.

    Science.gov (United States)

    Greening, David W; Nguyen, Hong P T; Elgass, Kirstin; Simpson, Richard J; Salamonsen, Lois A

    2016-02-01

    Embryo implantation into receptive endometrium requires synergistic endometrial-blastocyst interactions within the uterine cavity and is essential for establishing pregnancy. We demonstrate that exosomes (40-150 nm nanovesicles) released from endometrial epithelial cells are an important component of these interactions. We defined the proteome of purified endometrial epithelial-derived exosomes (Exos) influenced by menstrual cycle hormones estrogen (E; proliferative phase) and estrogen plus progesterone (EP; receptive phase) and examined their potential to modify trophoblast function. E-/EP-Exos were uniquely enriched with 254 and 126 proteins, respectively, with 35% newly identified proteins not previously reported in exosome databases. Importantly, EP-Exos protein cargo was related to fundamental changes in implantation: adhesion, migration, invasion, and extracellular matrix remodeling. These findings from hormonally treated ECC1 endometrial cancer cells were validated in human primary uterine epithelial cell-derived exosomes. Functionally, exosomes were internalized by human trophoblast cells and enhanced their adhesive capacity, a response mediated partially through active focal adhesion kinase (FAK) signaling. Thus, exosomes contribute to the endometrial-embryo interactions within the human uterine microenvironment essential for successful implantation. © 2016 by the Society for the Study of Reproduction, Inc.

  7. Secondary Release of Exosomes From Astrocytes Contributes to the Increase in Neural Plasticity and Improvement of Functional Recovery After Stroke in Rats Treated With Exosomes Harvested From MicroRNA 133b-Overexpressing Multipotent Mesenchymal Stromal Cells.

    Science.gov (United States)

    Xin, Hongqi; Wang, Fengjie; Li, Yanfeng; Lu, Qing-E; Cheung, Wing Lee; Zhang, Yi; Zhang, Zheng Gang; Chopp, Michael

    2017-02-16

    We previously demonstrated that multipotent mesenchymal stromal cells (MSCs) that overexpress microRNA 133b (miR-133b) significantly improve functional recovery in rats subjected to middle cerebral artery occlusion (MCAO) compared with naive MSCs and that exosomes generated from naive MSCs mediate the therapeutic benefits of MSC therapy for stroke. Here we investigated whether exosomes isolated from miR-133b-overexpressing MSCs (Ex-miR-133b+) exert amplified therapeutic effects. Rats subjected to 2 h of MCAO were intra-arterially injected with Ex-miR-133b+, exosomes from MSCs infected by blank vector (Ex-Con), or phosphate-buffered saline (PBS) and were sacrificed 28 days after MCAO. Compared with the PBS treatment, both exosome treatment groups exhibited significant improvement of functional recovery. Ex-miR-133b+ treatment significantly increased functional improvement and neurite remodeling/brain plasticity in the ischemic boundary area compared with the Ex-Con treatment. Treatment with Ex-miR-133b+ also significantly increased brain exosome content compared with Ex-Con treatment. To elucidate mechanisms underlying the enhanced therapeutic effects of Ex-miR-133b+, astrocytes cultured under oxygen- and glucose-deprived (OGD) conditions were incubated with exosomes harvested from naive MSCs (Ex-Naive), miR-133b downregulated MSCs (Ex-miR-133b-), and Ex-miR-133b+. Compared with the Ex-Naive treatment, Ex-miR-133b+ significantly increased exosomes released by OGD astrocytes, whereas Ex-miR-133b- significantly decreased the release. Also, exosomes harvested from OGD astrocytes treated with Ex-miR-133b+ significantly increased neurite branching and elongation of cultured cortical embryonic rat neurons compared with the exosomes from OGD astrocytes subjected to Ex-Con. Our data suggest that exosomes harvested from miR-133b-overexpressing MSCs improve neural plasticity and functional recovery after stroke with a contribution from a stimulated secondary release of

  8. Exosomes/tricalcium phosphate combination scaffolds can enhance bone regeneration by activating the PI3K/Akt signaling pathway.

    Science.gov (United States)

    Zhang, Jieyuan; Liu, Xiaolin; Li, Haiyan; Chen, Chunyuan; Hu, Bin; Niu, Xin; Li, Qing; Zhao, Bizeng; Xie, Zongping; Wang, Yang

    2016-09-20

    Recently, accumulating evidence has shown that exosomes, the naturally secreted nanocarriers of cells, can exert therapeutic effects in various disease models in the absence of parent cells. However, application of exosomes in bone defect repair and regeneration has been rarely reported, and little is known regarding their underlying mechanisms. Exosomes derived from human-induced pluripotent stem cell-derived mesenchymal stem cells (hiPS-MSC-Exos) were combined with tricalcium phosphate (β-TCP) to repair critical-sized calvarial bone defects, and the efficacy was assessed by histological examination. We evaluated the in vitro effects of hiPSC-MSC-Exos on the proliferation, migration, and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) by cell-counting, scratch assays, and qRT-PCR, respectively. Gene expression profiling and bioinformatics analyses were also used to identify the underlying mechanisms in the repair. We found that the exosome/β-TCP combination scaffolds could enhance osteogenesis as compared to pure β-TCP scaffolds. In vitro assays showed that the exosomes could release from β-TCP and could be internalized by hBMSCs. In addition, the internalization of exosomes into hBMSCs could profoundly enhance the proliferation, migration, and osteogenic differentiation of hBMSCs. Furthermore, gene expression profiling and bioinformatics analyses demonstrated that exosome/β-TCP combination scaffolds significantly altered the expression of a network of genes involved in the PI3K/Akt signaling pathway. Functional studies further confirmed that the PI3K/Akt signaling pathway was the critical mediator during the exosome-induced osteogenic responses of hBMSCs. We propose that the exosomes can enhance the osteoinductivity of β-TCP through activating the PI3K/Akt signaling pathway of hBMSCs, which means that the exosome/β-TCP combination scaffolds possess better osteogenesis activity than pure β-TCP scaffolds. These

  9. Heat Shock Protein HSP27 Secretion by Ovarian Cancer Cells Is Linked to Intracellular Expression Levels, Occurs Independently of the Endoplasmic Reticulum Pathway and HSP27’s Phosphorylation Status, and Is Mediated by Exosome Liberation

    Directory of Open Access Journals (Sweden)

    Matthias B. Stope

    2017-01-01

    Full Text Available The heat shock protein HSP27 has been correlated in ovarian cancer (OC patients with aggressiveness and chemoresistance and, therefore, represents a promising potential biomarker for OC diagnosis, prognosis, and treatment response. Notably, secretion of soluble HSP27 has been described by a few cell types and may take place as well in OC cells. Therefore, we studied HSP27 secretion mechanisms under diverse cellular conditions in an OC cell model system. Secretion of HSP27 was characterized after overexpression of HSP27 by transfected plasmids and after heat shock. Intra- and extracellular HSP27 amounts were assessed by Western blotting and ELISA. Protein secretion was blocked by brefeldin A and the impact of the HSP27 phosphorylation status was analyzed overexpressing HSP27 phosphomutants. The present study demonstrated that HSP27 secretion by OVCAR-3 and SK-OV-3 cells depends on intracellular HSP27 concentrations. Moreover, HSP27 secretion is independent of the endoplasmic reticulum secretory pathway and HSP27 phosphorylation. Notably, analysis of OC cell-born exosomes not only confirmed the concentration-dependent correlation of HSP27 expression and secretion but also demonstrated a concentration-dependent incorporation of HSP27 protein into exosomes. Thus, secreted HSP27 may become more important as an extracellular factor which controls the tumor microenvironment and might be a noninvasive biomarker.

  10. Exosomes: secreted vesicles and intercellular communications

    OpenAIRE

    Théry, Clotilde

    2011-01-01

    Exosomes are small membrane vesicles of endocytic origin secreted by most cell types, and are thought to play important roles in intercellular communications. Although exosomes were originally described in 1983, interest in these vesicles has really increased dramatically in the last 3 years, after the finding that they contain mRNA and microRNA. This discovery sparked renewed interest for the general field of membrane vesicles involved in intercellular communications, and research on these s...

  11. Exosomes in tumor microenvironment: novel transporters and biomarkers.

    Science.gov (United States)

    Wang, Zhen; Chen, Jun-Qiang; Liu, Jin-Lu; Tian, Lei

    2016-10-19

    Tumor microenvironment (TME) plays an integral part in the biology of cancer, participating in tumor initiation, progression, and response to therapy. Exosome is an important part of TME. Exosomes are small vesicles formed in vesicular bodies with a diameter of 30-100 nm and a classic "cup" or "dish" morphology. They can contain microRNAs, mRNAs, DNA fragments and proteins, which are shuttled from a donor cell to recipient cells. Exosomes secreted from tumor cells are called tumor-derived (TD) exosomes. There is emerging evidence that TD exosomes can construct a fertile environment to support tumor proliferation, angiogenesis, invasion and premetastatic niche preparation. TD exosomes also may facilitate tumor growth and metastasis by inhibiting immune surveillance and by increasing chemoresistance via removal of chemotherapeutic drugs. Therefore, TD-exosomes might be potential targets for therapeutic interventions via their modification or removal. For example, exosomes can serve as specific delivery vehicles to tumors of drugs, small molecules, or agents of prevention and gene therapy. Furthermore, the biomarkers detected in exosomes of biological fluids imply a potential for exosomes in the early detection and diagnosis, prediction of therapeutic efficacy, and determining prognosis of cancer. Although exosomes may serve as cancer biomarkers and aid in the treatment of cancer, we have a long way to go before we can further enhance the anti-tumor therapy of exosomes and develop exosome-based cancer diagnostic and therapeutic strategies.

  12. Dendritic cells release HLA-B-associated transcript-3 positive exosomes to regulate natural killer function.

    Directory of Open Access Journals (Sweden)

    Venkateswara Rao Simhadri

    Full Text Available NKp30, a natural cytotoxicity receptor expressed on NK cells is critically involved in direct cytotoxicity against various tumor cells and directs both maturation and selective killing of dendritic cells. Recently the intracellular protein BAT3, which is involved in DNA damage induced apoptosis, was identified as a ligand for NKp30. However, the mechanisms underlying the exposure of the intracellular ligand BAT3 to surface NKp30 and its role in NK-DC cross talk remained elusive. Electron microscopy and flow cytometry demonstrate that exosomes released from 293T cells and iDCs express BAT3 on the surface and are recognized by NKp30-Ig. Overexpression and depletion of BAT3 in 293T cells directly correlates with the exosomal expression level and the activation of NK cell-mediated cytokine release. Furthermore, the NKp30-mediated NK/DC cross talk resulting either in iDC killing or maturation was BAT3-dependent. Taken together this puts forward a new model for the activation of NK cells through intracellular signals that are released via exosomes from accessory cells. The manipulation of the exosomal regulation may offer a novel strategy to induce tumor immunity or inhibit autoimmune diseases caused by NK cell-activation.

  13. Exosomes in the Pathology of Neurodegenerative Diseases.

    Science.gov (United States)

    Howitt, Jason; Hill, Andrew F

    2016-12-23

    More than 30 years ago, two unexpected findings were discovered that challenged conventional thinking in biology. The first was the identification of a misfolded protein with transmissible properties associated with a group of neurodegenerative diseases known as transmissible spongiform encephalopathies. The second was the discovery of a new pathway used for the extracellular release of biomolecules, including extracellular vesicles called exosomes. Two decades later, the convergence of these pathways was shown when exosomes were found to play a significant role in both the transmission and propagation of protein aggregates in disease. Recent research has now revealed that the majority of proteins involved in neurodegenerative diseases are transported in exosomes, and that external stresses due to age-related impairment of protein quality control mechanisms can promote the transcellular flux of these proteins in exosomes. Significantly, exosomes provide an environment that can induce the conformational conversion of native proteins into aggregates that can be transmitted to otherwise aggregate-free cells in the brain. Here we review the current roles of exosomes in the pathology of neurodegenerative diseases. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. Exploring the RNA landscape of endothelial exosomes.

    Science.gov (United States)

    Perez-Boza, Jennifer; Lion, Michelle; Struman, Ingrid

    2017-12-27

    Exosomes are small extracellular vesicles of around 100nm of diameter produced by most cell types. These vesicles carry nucleic acids, proteins, lipids and other biomolecules and function as carriers of biological information in processes of extracellular communication. The content of exosomes is regulated by the external and internal microenvironment of the parent cell, but the intrinsic mechanisms of loading of molecules into exosomes is still not completely elucidated. In this study, by the use of next generation sequencing we have characterized in depth the RNA composition of healthy endothelial cells and exosomes and provided an accurate profile of the different coding and non-coding RNA species found per compartment. We have also discovered a set of unique genes preferentially included (or excluded) into vesicles. Moreover, after studying the enrichment of RNA motifs in the genes unequally distributed between cells and exosomes, we have detected a set of enriched sequences for several classes of RNA. In conclusion, our results provide the basis to study the involvement of RNA-binding proteins capable to recognize RNA sequences and their role in the export of RNAs into exosomes. Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  15. Exosomes isolated from sera of mice fed Lactobacillus strains affect inflammatory cytokine production in macrophages in vitro.

    Science.gov (United States)

    Aoki-Yoshida, Ayako; Saito, Shinichi; Tsuruta, Takeshi; Ohsumi, Arisa; Tsunoda, Hinako; Sonoyama, Kei

    2017-07-22

    Orally administered Lactobacillus strains, including L. plantarum No.14 and L. rhamnosus GG, reportedly reduce inflammatory cytokine production in mice. The present study tested our idea that circulating exosomes mediate the action of Lactobacillus strains. The lipopolysaccharide-induced production of TNF-α and IL-6 in vitro was attenuated in peritoneal exudate cells (PECs) isolated from C57BL/6N mice that had been fed L. plantarum No.14. When PECs were cultured for 24 h with exosomes isolated from the serum of mice fed L. plantarum No.14 or L. rhamnosus GG, accumulation of both TNF-α and of the corresponding mRNA was lowered. Growth in the presence of these exosomes also decreased the production of TNF-α and IL-6 by the murine macrophage cell line RAW264.7. In contrast, supplementation with exosome-depleted serum of mice fed L. plantarum No.14 or L. rhamnosus GG failed to affect the production of TNF-α and IL-6 by RAW264.7 cells. When PECs and RAW264.7 cells were cultured for 24 h with PKH67-labeled exosomes isolated from murine serum, fluorescent signal was observed inside the cells, suggesting that these cells incorporate serum exosomes. We propose that the anti-inflammatory activity of orally administered L. plantarum No.14 and L. rhamnosus GG is mediated, at least in part, by circulating exosomes. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Vaccinia virus induces rapid necrosis in keratinocytes by a STAT3-dependent mechanism.

    Directory of Open Access Journals (Sweden)

    Yong He

    Full Text Available Humans with a dominant negative mutation in STAT3 are susceptible to severe skin infections, suggesting an essential role for STAT3 signaling in defense against cutaneous pathogens.To focus on innate antiviral defenses in keratinocytes, we used a standard model of cutaneous infection of severe combined immunodeficient mice with the current smallpox vaccine, ACAM-2000. In parallel, early events post-infection with the smallpox vaccine ACAM-2000 were investigated in cultured keratinocytes of human and mouse origin.Mice treated topically with a STAT3 inhibitor (Stattic developed larger vaccinia lesions with higher virus titers and died more rapidly than untreated controls. Cultured human and murine keratinocytes infected with ACAM-2000 underwent rapid necrosis, but when treated with Stattic or with inhibitors of RIP1 kinase or caspase-1, they survived longer, produced higher titers of virus, and showed reduced activation of type I interferon responses and inflammatory cytokines release. Treatment with inhibitors of RIP1 kinase and STAT3, but not caspase-1, also reduced the inflammatory response of keratinocytes to TLR ligands. Vaccinia growth properties in Vero cells, which are known to be defective in some antiviral responses, were unaffected by inhibition of RIP1K, caspase-1, or STAT3.Our findings indicate that keratinocytes suppress the replication and spread of vaccinia virus by undergoing rapid programmed cell death, in a process requiring STAT3. These data offer a new framework for understanding susceptibility to skin infection in patients with STAT3 mutations. Interventions which promote prompt necroptosis/pyroptosis of infected keratinocytes may reduce risks associated with vaccination with live vaccinia virus.

  17. Leptin Protects Host Cells from Entamoeba histolytica Cytotoxicity by a STAT3-Dependent Mechanism

    Science.gov (United States)

    Verkerke, Hans P.; Paul, Shom N.; Mackey, Aaron J.; Petri, William A.

    2012-01-01

    The adipocytokine leptin links nutritional status to immune function. Leptin signaling protects from amebiasis, but the molecular mechanism is not understood. We developed an in vitro model of ameba-host cell interaction to test the hypothesis that leptin prevents ameba-induced apoptosis in host epithelial cells. We demonstrated that activation of mammalian leptin signaling increased cellular resistance to amebic cytotoxicity, including caspase-3 activation. Exogenous expression of the leptin receptor conferred resistance in susceptible cells, and leptin stimulation enhanced protection. A series of leptin receptor signaling mutants showed that resistance to amebic cytotoxicity was dependent on activation of STAT3 but not the Src homology-2 domain-containing tyrosine phosphatase (SHP-2) or STAT5. A common polymorphism in the leptin receptor (Q223R) that increases susceptibility to amebiasis in humans and mice was found to increase susceptibility to amebic cytotoxicity in single cells. The Q223R polymorphism also decreased leptin-dependent STAT3 activation by 21% relative to that of the wild-type (WT) receptor (P = 0.035), consistent with a central role of STAT3 signaling in protection. A subset of genes uniquely regulated by STAT3 in response to leptin was identified. Most notable were the TRIB1 and suppressor of cytokine signaling 3 (SOCS3) genes, which have opposing roles in the regulation of apoptosis. Overall apoptotic genes were highly enriched in this gene set (P leptin regulation of host apoptotic genes via STAT3 is responsible for protection. This is the first demonstration of a mammalian signaling pathway that restricts amebic pathogenesis and represents an important advance in our mechanistic understanding of how leptin links nutrition and susceptibility to infection. PMID:22331430

  18. The role of exosomes and miRNAs in drug-resistance of cancer cells.

    Science.gov (United States)

    Bach, Duc-Hiep; Hong, Ji-Young; Park, Hyen Joo; Lee, Sang Kook

    2017-07-15

    Chemotherapy, one of the principal approaches for cancer patients, plays a crucial role in controlling tumor progression. Clinically, tumors reveal a satisfactory response following the first exposure to the chemotherapeutic drugs in treatment. However, most tumors sooner or later become resistant to even chemically unrelated anticancer agents after repeated treatment. The reduced drug accumulation in tumor cells is considered one of the significant mechanisms by decreasing drug permeability and/or increasing active efflux (pumping out) of the drugs across the cell membrane. The mechanisms of treatment failure of chemotherapeutic drugs have been investigated, including drug efflux, which is mediated by extracellular vesicles (EVs). Exosomes, a subset of EVs with a size range of 40-150 nm and a lipid bilayer membrane, can be released by all cell types. They mediate specific cell-to-cell interactions and activate signaling pathways in cells they either fuse with or interact with, including cancer cells. Exosomal RNAs are heterogeneous in size but enriched in small RNAs, such as miRNAs. In the primary tumor microenvironment, cancer-secreted exosomes and miRNAs can be internalized by other cell types. MiRNAs loaded in these exosomes might be transferred to recipient niche cells to exert genome-wide regulation of gene expression. How exosomal miRNAs contribute to the development of drug resistance in the context of the tumor microenvironment has not been fully described. In this review, we will highlight recent studies regarding EV-mediated microRNA delivery in formatting drug resistance. We also suggest the use of EVs as an advancing method in antiresistance treatment. © 2017 UICC.

  19. Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment

    Science.gov (United States)

    Xiao, Deyi; Barry, Samantha; Kmetz, Daniel; Egger, Michael; Pan, Jianmin; Rai, Shesh N; Qu, Jifu; McMasters, Kelly M.; Hao, Hongying

    2016-01-01

    The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma. PMID:27063098

  20. Melanoma cell-derived exosomes promote epithelial-mesenchymal transition in primary melanocytes through paracrine/autocrine signaling in the tumor microenvironment.

    Science.gov (United States)

    Xiao, Deyi; Barry, Samantha; Kmetz, Daniel; Egger, Michael; Pan, Jianmin; Rai, Shesh N; Qu, Jifu; McMasters, Kelly M; Hao, Hongying

    2016-07-01

    The tumor microenvironment is abundant with exosomes that are secreted by the cancer cells themselves. Exosomes are nanosized, organelle-like membranous structures that are increasingly being recognized as major contributors in the progression of malignant neoplasms. A critical element in melanoma progression is its propensity to metastasize, but little is known about how melanoma cell-derived exosomes modulate the microenvironment to optimize conditions for tumor progression and metastasis. Here, we provide evidence that melanoma cell-derived exosomes promote phenotype switching in primary melanocytes through paracrine/autocrine signaling. We found that the mitogen-activated protein kinase (MAPK) signaling pathway was activated during the exosome-mediated epithelial-to-mesenchymal transition (EMT)-resembling process, which promotes metastasis. Let-7i, an miRNA modulator of EMT, was also involved in this process. We further defined two other miRNA modulators of EMT (miR-191 and let-7a) in serum exosomes for differentiating stage I melanoma patients from non-melanoma subjects. These results provide the first strong molecular evidence that melanoma cell-derived exosomes promote the EMT-resembling process in the tumor microenvironment. Thus, novel strategies targeting EMT and modulating the tumor microenvironment may emerge as important approaches for the treatment of metastatic melanoma. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. Plasmalogen enrichment in exosomes secreted by a nematode parasite versus those derived from its mouse host: implications for exosome stability and biology

    Directory of Open Access Journals (Sweden)

    Fabio Simbari

    2016-07-01

    Full Text Available Extracellular vesicles (EVs mediate communication between cells and organisms across all 3 kingdoms of life. Several reports have demonstrated that EVs can transfer molecules between phylogenetically diverse species and can be used by parasites to alter the properties of the host environment. Whilst the concept of vesicle secretion and uptake is broad reaching, the molecular composition of these complexes is expected to be diverse based on the physiology and environmental niche of different organisms. Exosomes are one class of EVs originally defined based on their endocytic origin, as these derive from multivesicular bodies that then fuse with the plasma membrane releasing them into the extracellular environment. The term exosome has also been used to describe any small EVs recovered by high-speed ultracentrifugation, irrespective of origin since this is not always well characterized. Here, we use comparative global lipidomic analysis to examine the composition of EVs, which we term exosomes, that are secreted by the gastrointestinal nematode, Heligmosomoides polygyrus, in relation to exosomes secreted by cells of its murine host. Ultra-performance liquid chromatography – tandem mass spectrometry (UPLC-MS/MS analysis reveals a 9- to 62-fold enrichment of plasmalogens, as well as other classes of ether glycerophospholipids, along with a relative lack of cholesterol and sphingomyelin (SM in the nematode exosomes compared with those secreted by murine cells. Biophysical analyses of the membrane dynamics of these exosomes demonstrate increased rigidity in those from the nematode, and parallel studies with synthetic vesicles support a role of plasmalogens in stabilizing the membrane structure. These results suggest that nematodes can maintain exosome membrane structure and integrity through increased plasmalogens, compensating for diminished levels of other lipids, including cholesterol and SM. This work also illuminates the prevalence of

  2. Micro RNA in Exosomes from HIV-Infected Macrophages.

    Science.gov (United States)

    Roth, William W; Huang, Ming Bo; Addae Konadu, Kateena; Powell, Michael D; Bond, Vincent C

    2015-12-22

    Exosomes are small membrane-bound vesicles secreted by cells that function to shuttle RNA and proteins between cells. To examine the role of exosomal micro RNA (miRNA) during the early stage of HIV-1 infection we characterized miRNA in exosomes from HIV-infected macrophages, compared with exosomes from non-infected macrophages. Primary human monocytes from uninfected donors were differentiated to macrophages (MDM) which were either mock-infected or infected with the macrophage-tropic HIV-1 BaL strain. Exosomes were recovered from culture media and separated from virus particles by centrifugation on iodixanol density gradients. The low molecular weight RNA fraction was prepared from purified exosomes. After pre-amplification, RNA was hybridized to microarrays containing probes for 1200 miRNA species of known and unknown function. We observed 48 miRNA species in both infected and uninfected MDM exosomes. Additionally, 38 miRNAs were present in infected-cell exosomes but not uninfected-cell exosomes. Of these, 13 miRNAs were upregulated in exosomes from HIV-infected cells, including 4 miRNA species that were increased by more than 10-fold. Though numerous miRNA species have been identified in HIV-infected cells, relatively little is known about miRNA content in exosomes from these cells. In the future, we plan to investigate whether the upregulated miRNA species we identified are increased in exosomes from HIV-1-positive patients.

  3. Interactions between Exosomes from Breast Cancer Cells and Primary Mammary Epithelial Cells Leads to Generation of Reactive Oxygen Species Which Induce DNA Damage Response, Stabilization of p53 and Autophagy in Epithelial Cells

    Science.gov (United States)

    Dutta, Sujoy; Warshall, Case; Bandyopadhyay, Chirosree; Dutta, Dipanjan; Chandran, Bala

    2014-01-01

    Exosomes are nanovesicles originating from multivesicular bodies and are released by all cell types. They contain proteins, lipids, microRNAs, mRNAs and DNA fragments, which act as mediators of intercellular communications by inducing phenotypic changes in recipient cells. Tumor-derived exosomes have been shown to play critical roles in different stages of tumor development and metastasis of almost all types of cancer. One of the ways by which exosomes affect tumorigenesis is to manipulate the tumor microenvironments to create tumor permissive “niches”. Whether breast cancer cell secreted exosomes manipulate epithelial cells of the mammary duct to facilitate tumor development is not known. To address whether and how breast cancer cell secreted exosomes manipulate ductal epithelial cells we studied the interactions between exosomes isolated from conditioned media of 3 different breast cancer cell lines (MDA-MB-231, T47DA18 and MCF7), representing three different types of breast carcinomas, and normal human primary mammary epithelial cells (HMECs). Our studies show that exosomes released by breast cancer cell lines are taken up by HMECs, resulting in the induction of reactive oxygen species (ROS) and autophagy. Inhibition of ROS by N-acetyl-L-cysteine (NAC) led to abrogation of autophagy. HMEC-exosome interactions also induced the phosphorylation of ATM, H2AX and Chk1 indicating the induction of DNA damage repair (DDR) responses. Under these conditions, phosphorylation of p53 at serine 15 was also observed. Both DDR responses and phosphorylation of p53 induced by HMEC-exosome interactions were also inhibited by NAC. Furthermore, exosome induced autophagic HMECs were found to release breast cancer cell growth promoting factors. Taken together, our results suggest novel mechanisms by which breast cancer cell secreted exosomes manipulate HMECs to create a tumor permissive microenvironment. PMID:24831807

  4. Exosomes and autophagy: coordinated mechanisms for the maintenance of cellular fitness

    Directory of Open Access Journals (Sweden)

    Francesc eBaixauli

    2014-08-01

    Full Text Available Conditions resulting from loss of cellular homeostasis; including oxidative stress, inflammation, protein aggregation, endoplasmic reticulum stress, metabolic stress and perturbation of mitochondrial function, are common to many pathological disorders and contribute to aging. Cells face these stress situations by engaging quality control mechanisms aimed to restore cellular homeostasis and preserve cell viability. Among them, the autophagy-lysosome pathway mediates the specific degradation of damaged proteins and organelles, and its proper function is related to cellular protection and increased life span in many model organisms. Besides autophagy, increasing evidence underscores a role for exosomes in the selective secretion of harmful/damaged proteins and RNAs and thus, in the maintenance of cellular fitness. In this perspective article, we discuss the emerging function of exosomes as means of alleviating intracellular stress conditions, and how secretion of harmful or unwanted material in exosomes, in coordination with the autophagy-lysosomal pathway, is essential to preserve intracellular protein and RNA homeostasis. Finally, we provide an overview about the consequences of the spreading of the exosome content in physiological

  5. Tumor-derived exosomes enhance invasion and metastasis of salivary adenoid cystic carcinoma cells.

    Science.gov (United States)

    Hou, Jin; Wang, Fangyuan; Liu, Xiaohao; Song, Mengyang; Yin, Xuemin

    2018-02-01

    Tumor-derived exosomes (TDE) have been shown to participate in different steps of the dissemination of cancer cells. However, the role of salivary adenoid cystic carcinoma-derived (SACC-derived) exosomes had not been documented in SACC. The study aims to explore the functions of SACC-derived TDE in SACC progression and investigate potential mechanisms. Salivary adenoid cystic carcinoma cell line SACC-83 was used to generate TDE. Afterward, SACC-83 or HUVECs were cocultured with or without TDE. Tumor migration, tumor invasion, and endothelial permeability were examined by wound healing assay, tumor invasion assay, endothelial permeability assay, and tumor cell transendothelial migration assay, respectively. Moreover, the expression levels of cell junction-related proteins were examined by qRT-PCR and Western blot. Salivary adenoid cystic carcinoma -83-derived exosomes were taken up by their host cells. Meanwhile, TDE increased migration and invasion capacity of SACC-83 cells and enhanced endothelial cell permeability. Furthermore, we demonstrated that the expression of cell junction-related proteins (Claudins and ZO-1) was downregulated, which is presumably involved in the TDE-mediated promotion of migration, invasion, and metastasis. The results suggested that SACC cell-derived exosomes were loaded with individual components that could enhance invasiveness and induce microenvironment changes, thus promoting SACC aggression. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  6. A visible and colorimetric aptasensor based on DNA-capped single-walled carbon nanotubes for detection of exosomes.

    Science.gov (United States)

    Xia, Yaokun; Liu, Mengmeng; Wang, Liangliang; Yan, An; He, Wenhui; Chen, Mei; Lan, Jianming; Xu, Jiaoxing; Guan, Lunhui; Chen, Jinghua

    2017-06-15

    Recently, many studies have shown the potential use of circulating exosomes as novel biomarkers for monitoring and predicting a number of complex diseases, including cancer. However, reliable and cost-effective detection of exosomes in routine clinical settings, still remain a difficult task, mainly due to the lack of adequately easy and fast assay platforms. Therefore, we demonstrate here the development of a visible and simple method for the detection of exosomes by integrating single-walled carbon nanotubes that being excellent water solubility (s-SWCNTs) and aptamer. Aptamers, specific to exosomes transmembrane protein CD63, are absorbed onto the surface of s-SWCNTs and improve the minic peroxidase activity of s-SWCNTs, which can efficiently catalyze H2O2-mediated oxidation of 3,3',5,5'-tetramethylbenzidine (TMB) and lead to a change from colorless to blue in solution. However, after adding exosomes, the aptamers are bound with CD63, leaving from the surface of s-SWCNTs through conformational changes, which results the color of solution from deep to moderate, and this can be observed by the naked eye and monitored by UV-vis spectrometry. Under optimal conditions, the linear range of exosomes is estimated to be 1.84×10(6) to 2.21×10(7) particles/μL with a detection of limit (LOD) of 5.2×10(5) particles/μL. Consequently, a visible and simple approach detecting exosomes is successfully constructed. Moreover, this proposed colorimetric aptasensor can be universally applicable for the detection of other targets by simple change the aptamer. Copyright © 2017 Elsevier B.V. All rights reserved.

  7. Exosomes from Glioma-Associated Mesenchymal Stem Cells Increase the Tumorigenicity of Glioma Stem-like Cells via Transfer of miR-1587.

    Science.gov (United States)

    Figueroa, Javier; Phillips, Lynette M; Shahar, Tal; Hossain, Anwar; Gumin, Joy; Kim, Hoon; Bean, Andrew J; Calin, George A; Fueyo, Juan; Walters, Edgar T; Kalluri, Raghu; Verhaak, Roel G; Lang, Frederick F

    2017-11-01

    Tumor-stromal communications impact tumorigenesis in ways that are incompletely understood. Here, we show that glioma-associated human mesenchymal stem cells (GA-hMSC), a newly identified stromal component of glioblastoma, release exosomes that increase the proliferation and clonogenicity of tumor-initiating glioma stem-like cells (GSC). This event leads to a significantly greater tumor burden and decreased host survival compared with untreated GSCs in orthotopic xenografts. Analysis of the exosomal content identified miR-1587 as a mediator of the exosomal effects on GSCs, in part via downregulation of the tumor-suppressive nuclear receptor corepressor NCOR1. Our results illuminate the tumor-supporting role for GA-hMSCs by identifying GA-hMSC-derived exosomes in the intercellular transfer of specific miRNA that enhance the aggressiveness of glioblastoma. Cancer Res; 77(21); 5808-19. ©2017 AACR. ©2017 American Association for Cancer Research.

  8. Exosome-associated hepatitis C virus in cell cultures and patient plasma

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Ziqing [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Zhang, Xiugen [Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States); Yu, Qigui [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); He, Johnny J., E-mail: johnny.he@unthsc.edu [Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis, IN 46202 (United States); Department of Cell Biology and Immunology, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2014-12-12

    Highlights: • HCV occurs in both exosome-free and exosome-associated forms. • Exosome-associated HCV is infectious and resistant to neutralizing antibodies. • More exosome-associated HCV than exosome-free HCV is present in patient plasma. - Abstract: Hepatitis C virus (HCV) infects its target cells in the form of cell-free viruses and through cell–cell contact. Here we report that HCV is associated with exosomes. Using highly purified exosomes and transmission electron microscopic imaging, we demonstrated that HCV occurred in both exosome-free and exosome-associated forms. Exosome-associated HCV was infectious and resistant to neutralization by an anti-HCV neutralizing antibody. There were more exosome-associated HCV than exosome-free HCV detected in the plasma of HCV-infected patients. These results suggest exosome-associated HCV as an alternative form for HCV infection and transmission.

  9. MEX3C interacts with adaptor-related protein complex 2 and involves in miR-451a exosomal sorting.

    Directory of Open Access Journals (Sweden)

    Pin Lu

    Full Text Available Some RNA species, especially microRNAs, are non-randomly sorted into exosomes, but how selectivity of RNA exosomal sorting is achieved is unknown. We found that all three variants of RNA-binding ubiquitin E3 ligase (MEX3C-MEX3C-1, MEX3C-2, and MEX3C-3 -interact with adaptor-related protein complex 2 (AP-2, a cargo adaptor in clathrin-mediated endocytosis. MEX3C's C-terminal RING finger domain and the hnRNP K homology (KH domain shared by the three MEX3C variants are both necessary for MEX3C/AP-2 interaction. MEX3C associates with the endolysosomal compartment through an endocytosis-like process. siRNA-mediated inhibition of the MEX3C or AP-2 complex substantially decreased exosomal but not cellular microRNA miR-451a expression. Exosomal sorting is ceramide-dependent but not ESCRT-dependent in microRNA miR-451a. That RNA-binding protein associates with membrane trafficking machinery, and that its involvement in exosomal microRNA expression, suggest the existence of a mechanism for specific recruiting of RNA molecules to endosomes for subsequent exosomal sorting.

  10. Trans-dissemination of exosomes from HIV-1-infected cells fosters both HIV-1 trans-infection in resting CD4+ T lymphocytes and reactivation of the HIV-1 reservoir.

    Science.gov (United States)

    Chiozzini, Chiara; Arenaccio, Claudia; Olivetta, Eleonora; Anticoli, Simona; Manfredi, Francesco; Ferrantelli, Flavia; d'Ettorre, Gabriella; Schietroma, Ivan; Andreotti, Mauro; Federico, Maurizio

    2017-09-01

    Intact HIV-1 and exosomes can be internalized by dendritic cells (DCs) through a common pathway leading to their transmission to CD4+ T lymphocytes by means of mechanisms defined as trans-infection and trans-dissemination, respectively. We previously reported that exosomes from HIV-1-infected cells activate both uninfected quiescent CD4+ T lymphocytes, which become permissive to HIV-1, and latently infected cells, with release of HIV-1 particles. However, nothing is known about the effects of trans-dissemination of exosomes produced by HIV-1-infected cells on uninfected or latently HIV-1-infected CD4+ T lymphocytes. Here, we report that trans-dissemination of exosomes from HIV-1-infected cells induces cell activation in resting CD4+ T lymphocytes, which appears stronger with mature than immature DCs. Using purified preparations of both HIV-1 and exosomes, we observed that mDC-mediated trans-dissemination of exosomes from HIV-1-infected cells to resting CD4+ T lymphocytes induces efficient trans-infection and HIV-1 expression in target cells. Most relevant, when both mDCs and CD4+ T lymphocytes were isolated from combination anti-retroviral therapy (ART)-treated HIV-1-infected patients, trans-dissemination of exosomes from HIV-1-infected cells led to HIV-1 reactivation from the viral reservoir. In sum, our data suggest a role of exosome trans-dissemination in both HIV-1 spread in the infected host and reactivation of the HIV-1 reservoir.

  11. MicroRNA-100 shuttled by mesenchymal stem cell-derived exosomes suppresses in vitro angiogenesis through modulating the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells.

    Science.gov (United States)

    Pakravan, Katayoon; Babashah, Sadegh; Sadeghizadeh, Majid; Mowla, Seyed Javad; Mossahebi-Mohammadi, Majid; Ataei, Farangis; Dana, Nasim; Javan, Mohammad

    2017-10-01

    Human mesenchymal stem cells (MSCs) have been shown to be involved in the formation and modulation of tumor stroma and in interacting with tumor cells, partly through their secretome. Exosomes are nano-sized intraluminal multi-vesicular bodies secreted by most types of cells and have been found to mediate intercellular communication through the transfer of genetic information via coding and non-coding RNAs to recipient cells. Since exosomes are considered as protective and enriched sources of shuttle microRNAs (miRNAs), we hypothesized that exosomal transfer of miRNAs from MSCs may affect tumor cell behavior, particularly angiogenesis. Exosomes derived from MSCs were isolated and characterized by scanning electron microscopy analyses, dynamic light scattering measurements, and Western blotting. Fold changes in miR-100 expression levels were calculated in exosomes and their corresponding donor cells by qRT-PCR. The effects of exosomal transfer of miR-100 from MSCs were assessed by qRT-PCR and Western blotting of the mTOR/HIF-1α/VEGF signaling axis in breast cancer cells. The quantification of secreted VEGF protein was determined by enzyme-linked immunosorbent assay. The putative paracrine effects of MSC-derived exosomes on tumor angiogenesis were explored by in vitro angiogenesis assays including endothelial cell proliferation, migration and tube formation assays. We found that MSC-derived exosomes induce a significant and dose-dependent decrease in the expression and secretion of vascular endothelial growth factor (VEGF) through modulating the mTOR/HIF-1α signaling axis in breast cancer-derived cells. We also found that miR-100 is enriched in MSC-derived exosomes and that its transfer to breast cancer-derived cells is associated with the down-regulation of VEGF in a time-dependent manner. The putative role of exosomal miR-100 transfer in regulating VEGF expression was substantiated by the ability of anti-miR-100 to rescue the inhibitory effects of MSC

  12. Cytosolic YB-1 and NSUN2 are the only proteins recognizing specific motifs present in mRNAs enriched in exosomes.

    Science.gov (United States)

    Kossinova, Olga A; Gopanenko, Alexander V; Tamkovich, Svetlana N; Krasheninina, Olga A; Tupikin, Alexey E; Kiseleva, Elena; Yanshina, Darya D; Malygin, Alexey A; Ven'yaminova, Alia G; Kabilov, Marsel R; Karpova, Galina G

    2017-06-01

    Exosomes, membranous vesicles secreted by various cells, are involved in intercellular communication and carry vast repertoires of RNAs and proteins. Processes mediating RNA sorting into exosomes are currently poorly understood. Using bioinformatics approaches, three structural motifs ACCAGCCU, CAGUGAGC and UAAUCCCA have been discovered as enriched in exosomal mRNAs and long noncoding RNAs. Here, utilizing short RNA hairpins, each containing one of the motifs, in a pull-down assay of cytosolic extract of human embryonic kidney 293 (HEK293) cells, we prove that multifunctional RNA-binding protein YB-1 specifically interacts with all three motifs, whereas methyltransferase NSUN2 recognizes only the motif CAGUGAGC. RNA hairpins other than those mentioned above pull out neither YB-1 nor NSUN2. Both these proteins are found in exosomes secreted by HEK293 cells. YB-1 for all that is detected as a form having a slightly higher electrophoretic mobility than that of YB-1 associated with the above RNA hairpins, assuming changes in posttranslational modifications of the protein during its transfer from cytoplasm into exosomes. Next generation sequencing of total exosomal RNA (eRNA) reveals a large representative set of RNA species, including mRNAs containing the above-mentioned motifs. The degree of enrichment in exosomes with this kind of mRNAs strongly depends on the locations of eRNA-specific motifs within the mRNA sequences. Altogether, our findings point to YB-1 and NSUN2 as possible mediators of the process of transfer of specific mRNAs into exosomes, allowing us to speculate on an involvement of these proteins in the mRNA sorting via the recognition of the above motifs. Copyright © 2017. Published by Elsevier B.V.

  13. Exosome nanotechnology: an emerging paradigm shift in drug delivery: exploitation of exosome nanovesicles for systemic in vivo delivery of RNAi heralds new horizons for drug delivery across biological barriers.

    Science.gov (United States)

    Lakhal, Samira; Wood, Matthew J A

    2011-10-01

    The demonstration that dendritic cell (DC)-derived exosomes can be exploited for targeted RNAi delivery to the brain after systemic injection provides the first proof-of-concept for the potential of these naturally occurring vesicles as vehicles of drug delivery. As well as being amenable to existing in vivo targeting strategies already in use for viruses and liposomes, this novel approach offers the added advantages of in vivo safety and low immunogenicity. Fulfilment of the potential of exosome delivery methods warrants a better understanding of their biology, as well as the development of novel production, characterisation, targeting and cargo-loading nanotechnologies. Ultimately, exosome-mediated drug delivery promises to overcome important challenges in the field of therapeutics, such as delivery of drugs across otherwise impermeable biological barriers, such as the blood brain barrier, and using patient-derived tissue as a source of individualised and biocompatible therapeutic drug delivery vehicles. Copyright © 2011 WILEY Periodicals, Inc.

  14. Heparanase activates the syndecan-syntenin-ALIX exosome pathway.

    Science.gov (United States)

    Roucourt, Bart; Meeussen, Sofie; Bao, Jie; Zimmermann, Pascale; David, Guido

    2015-04-01

    Exosomes are secreted vesicles of endosomal origin involved in signaling processes. We recently showed that the syndecan heparan sulfate proteoglycans control the biogenesis of exosomes through their interaction with syntenin-1 and the endosomal-sorting complex required for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-syntenin-ALIX exosome biogenesis pathway. We show that heparanase stimulates the exosomal secretion of syntenin-1, syndecan and certain other exosomal cargo, such as CD63, in a concentration-dependent manner. In contrast, exosomal CD9, CD81 and flotillin-1 are not affected. Conversely, reduction of endogenous heparanase reduces the secretion of syntenin-1-containing exosomes. The ability of heparanase to stimulate exosome production depends on syntenin-1 and ALIX. Syndecans, but not glypicans, support exosome biogenesis in heparanase-exposed cells. Finally, heparanase stimulates intraluminal budding of syndecan and syntenin-1 in endosomes, depending on the syntenin-ALIX interaction. Taken together, our findings identify heparanase as a modulator of the syndecan-syntenin-ALIX pathway, fostering endosomal membrane budding and the biogenesis of exosomes by trimming the heparan sulfate chains on syndecans. In addition, our data suggest that this mechanism controls the selection of specific cargo to exosomes.

  15. Pseudotyping exosomes for enhanced protein delivery in mammalian cells.

    Science.gov (United States)

    Meyer, Conary; Losacco, Joseph; Stickney, Zachary; Li, Lingxuan; Marriott, Gerard; Lu, Biao

    2017-01-01

    Exosomes are cell-derived nanovesicles that hold promise as living vehicles for intracellular delivery of therapeutics to mammalian cells. This potential, however, is undermined by the lack of effective methods to load exosomes with therapeutic proteins and to facilitate their uptake by target cells. Here, we demonstrate how a vesicular stomatitis virus glycoprotein (VSVG) can both load protein cargo onto exosomes and increase their delivery ability via a pseudotyping mechanism. By fusing a set of fluorescent and luminescent reporters with VSVG, we show the successful targeting and incorporation of VSVG fusions into exosomes by gene transfection and fluorescence tracking. We subsequently validate our system by live cell imaging of VSVG and its participation in endosomes/exosomes that are ultimately released from transfected HEK293 cells. We show that VSVG pseudotyping of exosomes does not affect the size or distributions of the exosomes, and both the full-length VSVG and the VSVG without the ectodomain are shown to integrate into the exosomal membrane, suggesting that the ectodomain is not required for protein loading. Finally, exosomes pseudotyped with full-length VSVG are internalized by multiple-recipient cell types to a greater degree compared to exosomes loaded with VSVG without the ectodomain, confirming a role of the ectodomain in cell tropism. In summary, our work introduces a new genetically encoded pseudotyping platform to load and enhance the intracellular delivery of therapeutic proteins via exosome-based vehicles to target cells.

  16. Comparative proteomics evaluation of plasma exosome isolation techniques and assessment of the stability of exosomes in normal human blood plasma.

    Science.gov (United States)

    Kalra, Hina; Adda, Christopher G; Liem, Michael; Ang, Ching-Seng; Mechler, Adam; Simpson, Richard J; Hulett, Mark D; Mathivanan, Suresh

    2013-11-01

    Exosomes are nanovesicles released by a variety of cells and are detected in body fluids including blood. Recent studies have highlighted the critical application of exosomes as personalized targeted drug delivery vehicles and as reservoirs of disease biomarkers. While these research applications have created significant interest and can be translated into practice, the stability of exosomes needs to be assessed and exosome isolation protocols from blood plasma need to be optimized. To optimize methods to isolate exosomes from blood plasma, we performed a comparative evaluation of three exosome isolation techniques (differential centrifugation coupled with ultracentrifugation, epithelial cell adhesion molecule immunoaffinity pull-down, and OptiPrep(TM) density gradient separation) using normal human plasma. Based on MS, Western blotting and microscopy results, we found that the OptiPrep(TM) density gradient method was superior in isolating pure exosomal populations, devoid of highly abundant plasma proteins. In addition, we assessed the stability of exosomes in plasma over 90 days under various storage conditions. Western blotting analysis using the exosomal marker, TSG101, revealed that exosomes are stable for 90 days. Interestingly, in the context of cellular uptake, the isolated exosomes were able to fuse with target cells revealing that they were indeed biologically active. © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Exosome-Based Cell-Cell Communication in the Tumor Microenvironment

    Directory of Open Access Journals (Sweden)

    Joana Maia

    2018-02-01

    Full Text Available Tumors are not isolated entities, but complex systemic networks involving cell-cell communication between transformed and non-transformed cells. The milieu created by tumor-associated cells may either support or halt tumor progression. In addition to cell-cell contact, cells communicate through secreted factors via a highly complex system involving characteristics such as ligand concentration, receptor expression and integration of diverse signaling pathways. Of these, extracellular vesicles, such as exosomes, are emerging as novel cell-cell communication mediators in physiological and pathological scenarios. Exosomes, membrane vesicles of endocytic origin released by all cells (both healthy and diseased, ranging in size from 30 to 150 nm, transport all the main biomolecules, including lipids, proteins, DNAs, messenger RNAs and microRNA, and perform intercellular transfer of components, locally and systemically. By acting not only in tumor cells, but also in tumor-associated cells such as fibroblasts, endothelium, leukocytes and progenitor cells, tumor- and non-tumor cells-derived exosomes have emerged as new players in tumor growth and invasion, tumor-associated angiogenesis, tissue inflammation and immunologic remodeling. In addition, due to their property of carrying molecules from their cell of origin to the peripheral circulation, exosomes have been increasingly studied as sources of tumor biomarkers in liquid biopsies. Here we review the current literature on the participation of exosomes in the communication between tumor and tumor-associated cells, highlighting the role of this process in the setup of tumor microenvironments that modulate tumor initiation and metastasis.

  18. Exosomes Derived From Natural Killer Cells Exert Therapeutic Effect in Melanoma.

    Science.gov (United States)

    Zhu, Liya; Kalimuthu, Senthilkumar; Gangadaran, Prakash; Oh, Ji Min; Lee, Ho Won; Baek, Se Hwan; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

    2017-01-01

    Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of NK cells. However, the characteristics and functions of exosomes derived from NK cells remain unknown. In this study, we explored NK cell-derived exosome-mediated antitumor effects against aggressive melanoma in vitro and in vivo. Methods: B16F10 cells were transfected with enhanced firefly luciferase (effluc) and thy1.1 genes, and thy1.1-positive cells were immunoselected using microbeads. The resulting B16F10/effluc cells were characterized using reverse transcriptase polymerase chain reaction (RT-PCR), western blotting, and luciferase activity assays. Exosomes derived from NK-92MI cells (NK-92 Exo) were isolated by ultracentrifugation and density gradient ultracentrifugation. NK-92 Exo were characterized by transmission electron microscopy and western blotting. We also performed an enzyme-linked immunosorbent assay to measure cytokines retained in NK-92 Exo cells. The in vitro cytotoxicity of NK-92 Exo against the cancer cells was determined using a bioluminescence imaging system (BLI) and CCK-8 assays. To investigate the possible side effects of NK-92 Exo on healthy cells, we also performed the BLI and CCK-8 assays using the human kidney Phoenix™-Ampho cell line. Flow cytometry and western blotting confirmed that NK-92 Exo induced apoptosis in the B16F10/effluc cells. In vivo, we used a B16F10/effluc cell xenograft model to detect the immunotherapeutic effect of NK-92 Exo. We injected NK-92 Exo into tumors, and tumor growth progression was monitored using the IVIS Lumina

  19. Proteomic profiling of exosomes: Current perspectives

    DEFF Research Database (Denmark)

    Simpson, Richard J; Jensen, Søren S; Lim, Justin W E

    2008-01-01

    distinct subsets of proteins that may be linked to cell-type associated functions. The secretion of exosomes by tumor cells and their implication in the transport and propagation of infectious cargo such as prions and retroviruses such as HIV suggest their participation in pathological situations...

  20. Isolation of nanoscale exosomes using viscoelastic effect

    Science.gov (United States)

    Hu, Guoqing; Liu, Chao

    2017-11-01

    Exosomes, molecular cargos secreted by almost all mammalian cells, are considered as promising biomarkers to identify many diseases including cancers. However, the small size of exosomes (30-200 nm) poses serious challenges on their isolation from the complex media containing a variety of extracellular vesicles (EVs) of different sizes, especially in small sample volumes. Here we develop a viscoelasticity-based microfluidic system to directly separate exosomes from cell culture media or serum in a continuous, size-dependent, and label-free manner. Using a small amount of biocompatible polymer as the additive into the media to control the viscoelastic forces exerted on EVs, we are able to achieve a high separation purity (>90%) and recovery (>80%) of exosomes. The size cutoff in viscoelasticity-based microfluidics can be easily controlled using different PEO concentrations. Based on this size-dependent viscoelastic separation strategy, we envision the handling of diverse nanoscale objects, such as gold nanoparticles, DNA origami structures, and quantum dots. This work was supported financially by National Natural Science Foundation of China (11572334, 91543125).

  1. HIV and mature dendritic cells: Trojan exosomes riding the Trojan horse?

    National Research Council Canada - National Science Library

    Izquierdo-Useros, Nuria; Naranjo-Gómez, Mar; Erkizia, Itziar; Puertas, Maria Carmen; Borràs, Francesc E; Blanco, Julià; Martinez-Picado, Javier

    2010-01-01

    ...). The Trojan exosome hypothesis proposes that retroviruses can take advantage of the cell-encoded intercellular vesicle traffic and exosome exchange pathway, moving between cells in the absence...

  2. Tumor-derived exosomes elicit tumor suppression in murine hepatocellular carcinoma models and humans in vitro.

    Science.gov (United States)

    Rao, Quan; Zuo, Bingfeng; Lu, Zhen; Gao, Xianjun; You, Abin; Wu, Chenxuan; Du, Zhi; Yin, HaiFang

    2016-08-01

    Hepatocellular carcinoma (HCC) remains a global challenge due to high morbidity and mortality rates and poor response to treatment. Immunotherapy, based on introduction of dendritic cells (DCs) activated by tumor cell lysates as antigens ex vivo, shows limited response rates in HCC patients. Here, we demonstrate that tumor cell-derived exosomes (TEXs), displaying an array of HCC antigens, can elicit a stronger immune response than cell lysates in vitro and in vivo. Significant tumor growth inhibition was achieved in ectopic and orthotopic HCC mice treated with TEX-pulsed DCs. Importantly, the tumor immune microenvironment was significantly improved in orthotopic HCC mice treated by TEX-pulsed DCs, demonstrated by increased numbers of T lymphocytes, elevated levels of interferon-γ, and decreased levels of interleukin-10 and tumor growth factor-β in tumor sites. As expected, T cells played an essential role in the TEX-pulsed DC-mediated immune response. Notably, exosomes from HCC cells not only promoted HCC-specific cytolysis but also provided cross-protective effects against pancreatic cancer cells. Moreover, HCC-specific cytolysis, elicited by DCs pulsed with human HepG2 cell-derived exosomes, was observed across different human HCC cells irrespective of human leukocyte antigen types. HCC TEXs can potently carry HCC antigens, trigger a strong DC-mediated immune response, and improve the HCC tumor microenvironment. (Hepatology 2016;64:456-472). © 2016 by the American Association for the Study of Liver Diseases.

  3. Functional Roles and Therapeutic Applications of Exosomes in Hepatocellular Carcinoma

    Directory of Open Access Journals (Sweden)

    Laura Santangelo

    2017-01-01

    Full Text Available Exosomes are important in intercellular communication. They assure the horizontal transfer of specific functional contents (i.e., proteins, lipids, RNA molecules, and circulating DNA from donor to recipient cells. Notably, tumor-derived exosomes (TDEs appear to be an important vehicle of specific signals in cancer, impacting on tumor growth and metastasis. Recent researches point to the characterization of exosomes in Hepatocellular Carcinoma (HCC, the major adult liver malignancy. In this review, we summarize current findings on HCC exosomes, focusing on the identification of noncoding RNAs as exosome-enriched functional regulators and new potential biomarkers. The great potential of exosomes in future HCC diagnostic and therapeutic approaches is underlined.

  4. Exosome: A Novel and Safer Therapeutic Refinement of Mesenchymal Stem Cell

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    Ronne Wee Yeh Yeo

    2013-01-01

    Full Text Available Mesenchymal stem cell (MSC has just been approved as the first “off-the-shelf” stem cell pharmaceutical drug with an anticipation of more approvals following completion of numerous rigorous clinical trials. Despite this progress, the rationale for MSC therapeutic efficacy remains tenuous and is increasingly rationalized on a secretion rather than differentiation mechanism. Recent studies identifying exosome as the secreted agent mediating MSC therapeutic efficacy could potentially reduce a cell-based drug to a safer biologic-based alternative. Here we review the development of MSC exosome as a potential first-in-class therapeutic, and the unique challenges in the manufacture and regulatory oversight of this new class of therapeutics.

  5. Smuggling gold nanoparticles across cell types - A new role for exosomes in gene silencing.

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    Roma-Rodrigues, Catarina; Pereira, Francisca; Alves de Matos, António P; Fernandes, Marta; Baptista, Pedro V; Fernandes, Alexandra R

    2017-05-01

    Once released to the extracellular space, exosomes enable the transfer of proteins, lipids and RNA between different cells, being able to modulate the recipient cells' phenotypes. Members of the Rab small GTP-binding protein family, such as RAB27A, are responsible for the coordination of several steps in vesicle trafficking, including budding, mobility, docking and fusion. The use of gold nanoparticles (AuNPs) for gene silencing is considered a cutting-edge technology. Here, AuNPs were functionalized with thiolated oligonucleotides anti-RAB27A (AuNP@PEG@anti-RAB27A) for selective silencing of the gene with a consequent decrease of exosomes´ release by MCF-7 and MDA-MB-453 cells. Furthermore, communication between tumor and normal cells was observed both in terms of alterations in c-Myc gene expression and transportation of the AuNPs, mediating gene silencing in secondary cells. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Human tumor-derived exosomes (TEX) regulate Treg functions via cell surface signaling rather than uptake mechanisms.

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    Muller, Laurent; Simms, Patricia; Hong, Chang-Sook; Nishimura, Michael I; Jackson, Edwin K; Watkins, Simon C; Whiteside, Theresa L

    2017-01-01

    Tumor-derived exosomes (TEX) are ubiquitously present in the tumor microenvironment and plasma of cancer patients. TEX carry a cargo of multiple stimulatory and inhibitory molecules and deliver them to recipient cells, serving as a communication network for the tumor. The mechanisms TEX use for delivering messages to recipient cells were evaluated using PKH26-labeled TEX produced by cultured human tumor cells, exosomes produced by dendritic cells-derived exosomes (DEX), or exosomes isolated from plasma of cancer patients (EXO). Human T-cell subsets, B cells, NK cells, and monocytes were co-incubated with TEX, DEX, or EXO and binding or internalization of labeled vesicles was evaluated by confocal microscopy and/or Amnis-based flow cytometry. Vesicle-induced Ca2+ influx in recipient T cells was monitored, and TEX-induced inosine production in Treg was determined by mass spectrometry. In contrast to B cells, NK cells or monocytes, conventional T cells did not internalize labeled vesicles. Minimal exosome uptake was only evident in Treg following prolonged co-incubation with TEX. All exosomes induced Ca2+ influx in T cells, with TEX and EXO isolated from cancer patients' plasma delivering the strongest, sustained signaling to Treg. Such sustained signaling resulted in the significant upregulation of the conversion of extracellular ATP to inosine (adenosine metabolite) by Treg, suggesting that TEX signaling could have functional consequences in these recipient cells. Thus, modulation of Treg suppressor functions by TEX is mediated by mechanisms dependent on cell surface signaling and does not require TEX internalization by recipient cells.

  7. Interplay between Autophagy, Exosomes and HIV-1 Associated Neurological Disorders: New Insights for Diagnosis and Therapeutic Applications

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    Chet Raj Ojha

    2017-07-01

    Full Text Available The autophagy–lysosomal pathway mediates a degradative process critical in the maintenance of cellular homeostasis as well as the preservation of proper organelle function by selective removal of damaged proteins and organelles. In some situations, cells remove unwanted or damaged proteins and RNAs through the release to the extracellular environment of exosomes. Since exosomes can be transferred from one cell to another, secretion of unwanted material to the extracellular environment in exosomes may have an impact, which can be beneficial or detrimental, in neighboring cells. Exosome secretion is under the influence of the autophagic system, and stimulation of autophagy can inhibit exosomal release and vice versa. Neurons are particularly vulnerable to degeneration, especially as the brain ages, and studies indicate that imbalances in genes regulating autophagy are a common feature of many neurodegenerative diseases. Cognitive and motor disease associated with severe dementia and neuronal damage is well-documented in the brains of HIV-infected individuals. Neurodegeneration seen in the brain in HIV-1 infection is associated with dysregulation of neuronal autophagy. In this paradigm, we herein provide an overview on the role of autophagy in HIV-associated neurodegenerative disease, focusing particularly on the effect of autophagy modulation on exosomal release of HIV particles and how this interplay impacts HIV infection in the brain. Specific autophagy–regulating agents are being considered for therapeutic treatment and prevention of a broad range of human diseases. Various therapeutic strategies for modulating specific stages of autophagy and the current state of drug development for this purpose are also evaluated.

  8. Exosomes in Tumor Microenvironment Influence Cancer Progression and Metastasis

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    Kahlert, Christoph; Kalluri, Raghu

    2013-01-01

    Exosomes are small membrane vesicles of endocytic origin with a size of 50 – 100 nm. They can contain microRNAs, mRNAs, DNA fragments and proteins, which are shuttled from a donar cell to recipient cells. Many different cell types including immune cells, mesenchymal cells and cancer cells release exosomes. There is emerging evidence that cancer-derived exosomes contribute to the recruitment and reprogramming of constituents associated with tumor environment. Here, we discuss different mechani...

  9. Exosomes from eosinophils autoregulate and promote eosinophil functions.

    Science.gov (United States)

    Cañas, José Antonio; Sastre, Beatriz; Mazzeo, Carla; Fernández-Nieto, Mar; Rodrigo-Muñoz, José Manuel; González-Guerra, Andrés; Izquierdo, Manuel; Barranco, Pilar; Quirce, Santiago; Sastre, Joaquín; Del Pozo, Victoria

    2017-05-01

    Eosinophils are able to secrete exosomes that have an undefined role in asthma pathogenesis. We hypothesized that exosomes released by eosinophils autoregulate and promote eosinophil function. Eosinophils of patients with asthma (n = 58) and healthy volunteers (n = 16) were purified from peripheral blood, and exosomes were isolated and quantified from eosinophils of the asthmatic and healthy populations. Apoptosis, adhesion, adhesion molecules expression, and migration assays were performed with eosinophils in the presence or absence of exosomes from healthy and asthmatic individuals. Reactive oxygen species (ROS) were evaluated by flow cytometry with an intracellular fluorescent probe and nitric oxide (NO) and a colorimetric kit. In addition, exosomal proteins were analyzed by mass spectrometry. Eosinophil-derived exosomes induced an increase in NO and ROS production on eosinophils. Moreover, exosomes could act as a chemotactic factor on eosinophils, and they produced an increase in cell adhesion, giving rise to a specific augmentation of adhesion molecules, such as ICAM-1 and integrin α2. Protein content between exosomes from healthy and asthmatic individuals seems to be similar in both groups. In conclusion, we found that exosomes from the eosinophils of patients with asthma could modify several specific eosinophil functions related to asthma pathogenesis and that they could contribute fundamentally to the development and maintenance of asthma. © Society for Leukocyte Biology.

  10. Exosomes in cancer theranostic: Diamonds in the rough.

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    Cordonnier, Marine; Chanteloup, Gaëtan; Isambert, Nicolas; Seigneuric, Renaud; Fumoleau, Pierre; Garrido, Carmen; Gobbo, Jessica

    2017-03-04

    During the last 10 years, exosomes, which are small vesicles of 50-200 nm diameter of endosomal origin, have aroused a great interest in the scientific and clinical community for their roles in intercellular communication in almost all physiological and pathological processes. Most cells can potentially release these nanovesicles that share with the parent cell a similar lipid bilayer with transmembrane proteins and a panel of enclosed soluble proteins such as heat shock proteins and genetic material, thus acting as potential nanoshuttles of biomarkers. Exosomes surface proteins allow their targeting and capture by recipient cells, while the exosomes' content can modify the physiological state of recipient cells. Tumor derived exosomes by interacting with other cells of the tumor microenvironment modulate tumor progression, angiogenic switch, metastasis, and immune escape. Targeting tumor-derived exosomes might be an interesting approach in cancer therapy. Furthermore, because a key issue to improve cancer patients' outcome relies on earlier cancer diagnosis (metastases, as opposed to the primary tumor, are responsible for most cancer deaths) exosomes have been put forward as promising biomarker candidates for cancer diagnosis and prognosis. This review summarizes the roles of exosomes in cancer and clinical interest, focusing on the importance of exosomal heat shock proteins (HSP). The challenges of clinical translation of HSP-exosomes as therapeutic targets and biomarkers for early cancer detection are also discussed.

  11. Exosomes: From Garbage Bins to Promising Therapeutic Targets.

    Science.gov (United States)

    H Rashed, Mohammed; Bayraktar, Emine; K Helal, Gouda; Abd-Ellah, Mohamed F; Amero, Paola; Chavez-Reyes, Arturo; Rodriguez-Aguayo, Cristian

    2017-03-02

    Intercellular communication via cell-released vesicles is a very important process for both normal and tumor cells. Cell communication may involve exosomes, small vesicles of endocytic origin that are released by all types of cells and are found in abundance in body fluids, including blood, saliva, urine, and breast milk. Exosomes have been shown to carry lipids, proteins, mRNAs, non-coding RNAs, and even DNA out of cells. They are more than simply molecular garbage bins, however, in that the molecules they carry can be taken up by other cells. Thus, exosomes transfer biological information to neighboring cells and through this cell-to-cell communication are involved not only in physiological functions such as cell-to-cell communication, but also in the pathogenesis of some diseases, including tumors and neurodegenerative conditions. Our increasing understanding of why cells release exosomes and their role in intercellular communication has revealed the very complex and sophisticated contribution of exosomes to health and disease. The aim of this review is to reveal the emerging roles of exosomes in normal and pathological conditions and describe the controversial biological role of exosomes, as it is now understood, in carcinogenesis. We also summarize what is known about exosome biogenesis, composition, functions, and pathways and discuss the potential clinical applications of exosomes, especially as biomarkers and novel therapeutic agents.

  12. Circulating osteocyte-derived exosomes contain miRNAs which are enriched in exosomes from MLO-Y4 cells.

    Science.gov (United States)

    Sato, Mari; Suzuki, Tomohide; Kawano, Mitsuoki; Tamura, Masato

    2017-02-01

    Signaling molecules produced by osteocytes have been proposed to serve as soluble factors that contribute to bone remodeling, as well as to homeostasis of other organs. However, to the best of our knowledge, there are currently no studies investigating the role of osteocyte-secreted exosomes. In the present study, ablation of osteocytes in mice [osteocyte-less (OL)] was used to examine the microRNA (miRNA) levels of plasma-circulating exosomes. In order to investigate the function of osteocyte-secreted exosomes, exosomes derived from MLO-Y4 cells were extracted and their miRNA expression levels were examined using miRNA array analysis and deep sequencing. Comparison of miRNA expression levels between plasma exosomes from OL mouse plasma and MLO-Y4-derived exosomes revealed that decreases in the number of miRNAs from exosomes circulating in the OL mouse plasma may be caused by a decrease in secretion of exosomes from osteocytes. These results suggest that osteocytes secrete exosomes containing characterized miRNAs and then circulate in the blood, and may thus transfer their components, including miRNAs, to recipient cells where they function as signaling molecules in other organs and/or tissues to regulate biological responses.

  13. Perturbations in the Urinary Exosome in Transplant Rejection

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    Sigdel, Tara K.; NG, Yolanda; Lee, Sangho; Nicora, Carrie D.; Qian, Weijun; Smith, Richard D.; Camp, David G.; Sarwal, Minnie M.

    2015-01-05

    Background: Urine exosomes, vesicles exocytosed into urine by all renal epithelial cell types, occur under normal physiologic and disease states. Exosome contents may mirror disease-specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed and for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery. Methods: Urine exosomes were isolated by centrifugal filtration from mid-stream, second morning void, urine samples collected from kidney transplant recipients with and without biopsy matched acute rejection. The proteomes of unfractionated whole urine (Uw) and urine exosomes (Uexo) underwent mass spectrometry-based quantitative proteomics analysis. The proteome data were analyzed for significant differential protein abundances in acute rejection (AR). Results: Identifications of 1018 and 349 proteins, Uw and Uexo fractions, respectively, demonstrated a 279 protein overlap between the two urinary compartments with 25%(70) of overlapping proteins unique to Uexoand represented membrane bound proteins (p=9.31e-7). Of 349 urine exosomal proteins identified in transplant patients 220 were not previously identified in the normal urine exosomal fraction. Uexo proteins (11), functioning in the inflammatory / stress response, were more abundant in patients with biopsy-confirmed acute rejection, 3 of which were exclusive to Uexo. Uexo AR-specific biomarkers (8) were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw. Conclusions: A rapid urinary exosome isolation method and quantitative measurement of enriched Uexo proteins was applied. Urine proteins specific to the exosomal fraction were detected either in unfractionated urine (at low abundances) or by Uexo fraction analysis. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were

  14. CD63 Regulates Epstein-Barr Virus LMP1 Exosomal Packaging, Enhancement of Vesicle Production, and Noncanonical NF-κB Signaling.

    Science.gov (United States)

    Hurwitz, Stephanie N; Nkosi, Dingani; Conlon, Meghan M; York, Sara B; Liu, Xia; Tremblay, Deanna C; Meckes, David G

    2017-03-01

    Latent membrane protein 1 (LMP1) is an Epstein-Barr virus (EBV)-encoded oncoprotein that is packaged into small extracellular vesicles (EVs) called exosomes. Trafficking of LMP1 into multivesicular bodies (MVBs) alters the content and function of exosomes. LMP1-modified exosomes enhance the growth, migration, and invasion of malignant cells, demonstrating the capacity to manipulate the tumor microenvironment and enhance the progression of EBV-associated cancers. Despite the growing evidence surrounding the significance of LMP1-modified exosomes in cancer, very little is understood about the mechanisms that orchestrate LMP1 incorporation into these vesicles. Recently, LMP1 was shown to be copurified with CD63, a conserved tetraspanin protein enriched in late endosomal and lysosomal compartments. Here, we demonstrate the importance of CD63 presence for exosomal packaging of LMP1. Nanoparticle tracking analysis and gradient purification revealed an increase in extracellular vesicle secretion and exosomal proteins following LMP1 expression. Immunoisolation of CD63-positive exosomes exhibited accumulation of LMP1 in this vesicle population. Functionally, CRISPR/Cas9 knockout of CD63 resulted in a reduction of LMP1-induced particle secretion. Furthermore, LMP1 packaging was severely impaired in CD63 knockout cells, concomitant with a disruption in the perinuclear localization of LMP1. Importantly, LMP1 trafficking to lipid rafts and activation of NF-κB and PI3K/Akt pathways remained intact following CD63 knockout, while mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and noncanonical NF-κB activation were observed to be increased. These results suggest that CD63 is a critical player in LMP1 exosomal trafficking and LMP1-mediated enhancement of exosome production and may play further roles in limiting downstream LMP1 signaling.IMPORTANCE EBV is a ubiquitous gamma herpesvirus linked to malignancies such as nasopharyngeal carcinoma

  15. Herpesviruses shape tumour microenvironment through exosomal transfer of viral microRNAs.

    Directory of Open Access Journals (Sweden)

    Ohad Yogev

    2017-08-01

    Full Text Available Metabolic changes within the cell and its niche affect cell fate and are involved in many diseases and disorders including cancer and viral infections. Kaposi's sarcoma-associated herpesvirus (KSHV is the etiological agent of Kaposi's sarcoma (KS. KSHV latently infected cells express only a subset of viral genes, mainly located within the latency-associated region, among them 12 microRNAs. Notably, these miRNAs are responsible for inducing the Warburg effect in infected cells. Here we identify a novel mechanism enabling KSHV to manipulate the metabolic nature of the tumour microenvironment. We demonstrate that KSHV infected cells specifically transfer the virus-encoded microRNAs to surrounding cells via exosomes. This flow of genetic information results in a metabolic shift toward aerobic glycolysis in the surrounding non-infected cells. Importantly, this exosome-mediated metabolic reprogramming of neighbouring cells supports the growth of infected cells, thereby contributing to viral fitness. Finally, our data show that this miRNA transfer-based regulation of cell metabolism is a general mechanism used by other herpesviruses, such as EBV, as well as for the transfer of non-viral onco-miRs. This exosome-based crosstalk provides viruses with a mechanism for non-infectious transfer of genetic material without production of new viral particles, which might expose them to the immune system. We suggest that viruses and cancer cells use this mechanism to shape a specific metabolic niche that will contribute to their fitness.

  16. Biodistribution and Delivery Efficiency of Unmodified Tumor-Derived Exosomes

    Science.gov (United States)

    Smyth, Tyson; Kullberg, Max; Malik, Noeen; Smith-Jones, Peter; Graner, Michael W.; Anchordoquy, Thomas J.

    2014-01-01

    The use of exosomes as a drug delivery vehicle has gained considerable interest. To establish if exosomes could be utilized effectively for drug delivery, a better understanding of their in vivo fate must be established. Through comparisons to liposomal formulations, which have been studied extensively for the last thirty years, we were able to make some comprehensive conclusions about the fate of unmodified tumor-derived exosomes in vivo. We observed a comparable rapid clearance and minimal tumor accumulation of intravenously-injected exosomes, PC:Chol liposomes, and liposomes formulated with the lipid extract of exosomes, suggesting the unique protein and lipid composition of exosomes does not appreciably impact exosomes’ rate of clearance and biodistribution. This rapid clearance along with minimal tumor accumulation of unmodified exosomes limits their use as an anti-cancer drug delivery vehicle; however, when delivered intratumorally, exosomes remained associated with tumor tissue to a significantly greater extent than PC:Chol liposomes. Furthermore, experiments utilizing mice with impaired adaptive or innate immune systems, revealed the significance of the innate immune system along with the complement protein C5 on exosomes’ rate of clearance. PMID:25523519

  17. Proteogenomic analysis reveals exosomes are more oncogenic than ectosomes

    Science.gov (United States)

    Liem, Michael; Fonseka, Pamali; Atukorala, Ishara; Ozcitti, Cemil; Mechler, Adam; Adda, Christopher G.; Ang, Ching-Seng; Mathivanan, Suresh

    2015-01-01

    Extracellular vesicles (EVs) include the exosomes (30-100 nm) that are produced through the endocytic pathway via the multivesicular bodies and the ectosomes (100-1000 nm) that are released through the budding of the plasma membrane. Despite the differences in the mode of biogenesis and size, reliable markers that can distinguish between exosomes and ectosomes are non-existent. Moreover, the precise functional differences between exosomes and ectosomes remains poorly characterised. Here, using label-free quantitative proteomics, we highlight proteins that could be exploited as markers to discriminate between exosomes and ectosomes. For the first time, a global proteogenomics analysis unveiled the secretion of mutant proteins that are implicated in cancer progression through tumor-derived EVs. Follow up integrated bioinformatics analysis highlighted the enrichment of oncogenic cargo in exosomes and ectosomes. Interestingly, exosomes induced significant cell proliferation and migration in recipient cells compared to ectosomes confirming the oncogenic nature of exosomes. These findings ascertain that cancer cells facilitate oncogenesis by the secretion of mutant and oncoproteins into the tumor microenvironment via exosomes and ectosomes. The integrative proteogenomics approach utilized in this study has the potential to identify disease biomarker candidates which can be later assayed in liquid biopsies obtained from cancer patients. PMID:25944692

  18. 3D plasmonic nanobowl platform for the study of exosomes in solution

    Science.gov (United States)

    Lee, Changwon; Carney, Randy P.; Hazari, Sidhartha; Smith, Zachary J.; Knudson, Alisha; Robertson, Christopher S.; Lam, Kit S.; Wachsmann-Hogiu, Sebastian

    2015-05-01

    Thin silver film coated nanobowl Surface Enhanced Raman Spectroscopy (SERS) substrates are used to capture exosomes in solution for SERS measurements that can provide biochemical analysis of intact and ruptured exosomes. Exosomes derived via Total Exosome Isolation Reagent (TEIR) as well as ultracentrifugation (UC) from the SKOV3 cell line were analyzed. Spectra of exosomes derived via TEIR are dominated by a signal characteristic for the TEIR kit that needs to be subtracted for all measurements. Differences in SERS spectra recorded at different times during the drying of the exosome solution are statistically analyzed with Principal Component Analysis (PCA). At the beginning of the drying process, SERS spectra of exosomes exhibit peaks characteristic for both lipids and proteins. Later on during the drying process, new SERS peaks develop, suggesting that the initially intact exosome ruptures over time. This time-dependent evolution of SERS peaks enables analysis of exosomal membrane contents and the contents inside the exosomes.

  19. Exosomes Derived from Breast Cancer Cells, Small Trojan Horses?

    Science.gov (United States)

    Villagrasa, Alejandro; Álvarez, Pablo Juan; Osuna, Antonio; Garrido, Jose Manuel; Aránega, Antonia; Rodríguez-Serrano, Fernando

    2014-12-01

    Exosomes are small extracellular vesicles secreted to the extracellular environment by several cell types, including tumor cells. It has been demonstrated that exosomes have an important role in intercellular communication, but they have recently been implicated in various tumor processes, including the oncogenic transformation of cells in the tumor microenvironment, tumor drug resistance, and the transport of tumor factors. Tumors appear to use exosomes to dialogue with and transform neighboring cells to create an ideal environment for their growth and expansion. On the other hand, the structure and function of exosomes may make them useful in cancer diagnosis and prognosis, because they contain molecules that could serve as biomarkers, including oncogenes, miRNAs, and certain proteins. They have the ability to travel via body fluids, from which they could be isolated and used to transport drugs to specific targets. This review aims to provide an update on the role of exosomes derived from breast cancer cells.

  20. MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis.

    NARCIS (Netherlands)

    Buschow, S.I.; Balkom, B.W.M. van; Aalberts, M.; Heck, A.J.R. van; Wauben, M.; Stoorvogel, W.

    2010-01-01

    Professional antigen-presenting cells secrete major histocompatibility complex class II (MHC II) carrying exosomes with unclear physiological function(s). Exosomes are first generated as the intraluminal vesicles (ILVs) of a specific type of multivesicular body, and are then secreted by fusion of

  1. Identification and proteomic analysis of osteoblast-derived exosomes

    Energy Technology Data Exchange (ETDEWEB)

    Ge, Min; Ke, Ronghu; Cai, Tianyi; Yang, Junyi; Mu, Xiongzheng, E-mail: cranio@vip.163.com

    2015-11-06

    Exosomes are nanometer-sized vesicles with the function of intercellular communication, and they are released by various cell types. To reveal the knowledge about the exosomes from osteoblast, and explore the potential functions of osteogenesis, we isolated microvesicles from supernatants of mouse Mc3t3 by ultracentrifugation, characterized exosomes by electron microscopy and immunoblotting and presented the protein profile by proteomic analysis. The result demonstrated that microvesicles were between 30 and 100 nm in diameter, round shape with cup-like concavity and expressed exosomal marker tumor susceptibility gene (TSG) 101 and flotillin (Flot) 1. We identified a total number of 1069 proteins among which 786 proteins overlap with ExoCarta database. Gene Oncology analysis indicated that exosomes mostly derived from plasma membrane and mainly involved in protein localization and intracellular signaling. The Ingenuity Pathway Analysis showed pathways are mostly involved in exosome biogenesis, formation, uptake and osteogenesis. Among the pathways, eukaryotic initiation factor 2 pathways played an important role in osteogenesis. Our study identified osteoblast-derived exosomes, unveiled the content of them, presented potential osteogenesis-related proteins and pathways and provided a rich proteomics data resource that will be valuable for further studies of the functions of individual proteins in bone diseases. - Highlights: • We for the first time identified exosomes from mouse osteoblast. • Osteoblasts-derived exosomes contain osteoblast peculiar proteins. • Proteins from osteoblasts-derived exosomes are intently involved in EIF2 pathway. • EIF2α from the EIF2 pathway plays an important role in osteogenesis.

  2. The Possible Role of Extravillous Trophoblast-Derived Exosomes on the Uterine Spiral Arterial Remodeling under Both Normal and Pathological Conditions

    Directory of Open Access Journals (Sweden)

    Carlos Salomon

    2014-01-01

    Full Text Available A tenet of contemporary obstetrics is that events that compromise placentation increase the risk of complications of pregnancy and contribute to poor pregnancy outcome. In particular, conditions that affect the invasion of placental cells and remodeling of uterine spiral arteries compromise placental function and the subsequent development of the fetus. Extravillous trophoblast cells (EVTs proliferate and migrate from the cytotrophoblast in the anchoring villi of the placenta and invade the maternal decidua and myometrium. These cells are localised with uterine uterine spiral arteries and are thought to induce vascular remodeling. A newly identified pathway by which EVTs may regulate vascular remodeling within the uterus is via the release of exosomes. Trophoblast cells release exosomes that mediate aspects of cell-to-cell communication. The aim of this brief commentary is to review the putative role of exosomes released from extravillous trophoblast cells in uterine spiral artery remodeling and, in particular, their role in the aetiology of preeclampsia. Placental exosomes may engage in local cell-to-cell communication between the cell constituents of the placenta and contiguous maternal tissues and/or distal interactions, involving the release of placental exosomes into biological fluids and their transport to a remote site of action.

  3. Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1.

    Science.gov (United States)

    Morel, Lydie; Regan, Melissa; Higashimori, Haruki; Ng, Seng Kah; Esau, Christine; Vidensky, Svetlana; Rothstein, Jeffrey; Yang, Yongjie

    2013-03-08

    Perisynaptic astrocytes express important glutamate transporters, especially excitatory amino acid transporter 2 (EAAT2, rodent analog GLT1) to regulate extracellular glutamate levels and modulate synaptic activation. In this study, we investigated an exciting new pathway, the exosome-mediated transfer of microRNA (in particular, miR-124a), in neuron-to-astrocyte signaling. Exosomes isolated from neuron-conditioned medium contain abundant microRNAs and small RNAs. These exosomes can be directly internalized into astrocytes and increase astrocyte miR-124a and GLT1 protein levels. Direct miR-124a transfection also significantly and selectively increases protein (but not mRNA) expression levels of GLT1 in cultured astrocytes. Consistent with our in vitro findings, intrastriatal injection of specific antisense against miR-124a into adult mice dramatically reduces GLT1 protein expression and glutamate uptake levels in striatum without reducing GLT1 mRNA levels. MiR-124a-mediated regulation of GLT1 expression appears to be indirect and is not mediated by its suppression of the putative GLT1 inhibitory ligand ephrinA3. Moreover, miR-124a is selectively reduced in the spinal cord tissue of end-stage SOD1 G93A mice, the mouse model of ALS. Subsequent exogenous delivery of miR-124a in vivo through stereotaxic injection significantly prevents further pathological loss of GLT1 proteins, as determined by GLT1 immunoreactivity in SOD1 G93A mice. Together, our study characterized a new neuron-to-astrocyte communication pathway and identified miRNAs that modulate GLT1 protein expression in astrocytes in vitro and in vivo.

  4. B lymphocytes and macrophages release cell membrane deposited C3-fragments on exosomes with T cell response-enhancing capacity.

    Science.gov (United States)

    Papp, Krisztián; Végh, Péter; Prechl, József; Kerekes, Krisztina; Kovács, János; Csikós, György; Bajtay, Zsuzsa; Erdei, Anna

    2008-04-01

    Recently exosomes have been shown to play important roles in several immune phenomena. These small vesicles contain MHC proteins along with co-stimulatory and adhesion molecules, and mediate antigen presentation to T cells. In the present study we show that upon incubation with autologous serum, murine macrophages and B cells--but not T lymphocytes--fix C3-fragments covalently to the cell membrane and release them on exosomes in a time dependent fashion. While in the case of human B lymphocytes CR2 has been shown to serve as the main C3b-acceptor site, here we clearly demonstrate that cells derived from CR1/2 KO animals also have the capacity to fix C3b covalently. This finding points to a major difference between human and murine systems, and suggests the existence of additional acceptor sites on the cell membrane. Here we show that C3-fragment containing exosomes derived from OVA loaded antigen presenting cells induce a significantly elevated T cell response in the presence of suboptimal antigen stimulus. These data reveal a novel function of cell surface-deposited C3-fragments and provide further evidence for the role of exosomes secreted by antigen presenting cells. Since fixation of C3b to plasma membranes can be substantial in the presence of pathogens; moreover tumor cells are also known to activate the complement system resulting in complement-deposition, C3-carrying exosomes released by these cells may play an important immunomodulatory role in vivo, as well.

  5. Cancer Cell-derived Exosomes Induce Mitogen-activated Protein Kinase-dependent Monocyte Survival by Transport of Functional Receptor Tyrosine Kinases*

    Science.gov (United States)

    Song, Xiao; Ding, Yanping; Liu, Gang; Yang, Xiao; Zhao, Ruifang; Zhang, Yinlong; Zhao, Xiao; Anderson, Gregory J.; Nie, Guangjun

    2016-01-01

    Tumor-associated macrophages (TAM) play pivotal roles in cancer initiation and progression. Monocytes, the precursors of TAMs, normally undergo spontaneous apoptosis within 2 days, but can subsist in the inflammatory tumor microenvironment for continuous survival and generation of sufficient TAMs. The mechanisms underlying tumor-driving monocyte survival remain obscure. Here we report that cancer cell-derived exosomes were crucial mediators for monocyte survival in the inflammatory niche. Analysis of the survival-promoting molecules in monocytes revealed that cancer cell-derived exosomes activated Ras and extracellular signal-regulated kinases in the mitogen-activated protein kinase (MAPK) pathway, resulting in the prevention of caspase cleavage. Phosphorylated receptor tyrosine kinases (RTKs), such as phosphorylated epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER-2), were abundantly expressed in cancer cell-derived exosomes. Knock-out of EGFR or/and HER-2, or alternatively, inhibitors against their phosphorylation significantly disturbed the exosome-mediated activation of the MAPK pathway, inhibition of caspase cleavage, and increase in survival rate in monocytes. Moreover, the deprived survival-stimulating activity of exosomes due to null expression of EGFR and HER-2 could be restored by activation of another RTK, insulin receptor. Overall, our study uncovered a mechanism of tumor-associated monocyte survival and demonstrated that cancer cell-derived exosomes can stimulate the MAPK pathway in monocytes through transport of functional RTKs, leading to inactivation of apoptosis-related caspases. This work provides insights into the long sought question on monocyte survival prior to formation of plentiful TAMs in the tumor microenvironment. PMID:26895960

  6. Exosomes: novel effectors of human platelet lysate activity.

    Science.gov (United States)

    Torreggiani, E; Perut, F; Roncuzzi, L; Zini, N; Baglìo, S R; Baldini, N

    2014-09-22

    Despite the popularity of platelet-rich plasma (PRP) and platelet lysate (PL) in orthopaedic practice, the mechanism of action and the effectiveness of these therapeutic tools are still controversial. So far, the activity of PRP and PL has been associated with different growth factors (GF) released during platelet degranulation. This study, for the first time, identifies exosomes, nanosized vesicles released in the extracellular compartment by a number of elements, including platelets, as one of the effectors of PL activity. Exosomes were isolated from human PL by differential ultracentrifugation, and analysed by electron microscopy and Western blotting. Bone marrow stromal cells (MSC) treated with three different exosome concentrations (0.6 μg, 5 μg and 50 μg) showed a significant, dose-dependent increase in cell proliferation and migration compared to the control. In addition, osteogenic differentiation assays demonstrated that exosome concentration differently affected the ability of MSC to deposit mineralised matrix. Finally, the analysis of exosome protein content revealed a higher amount of basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF-BB) and transforming growth factor beta 1 (TGF-β1) as compared to PL. In regards to RNA content, an enrichment of small RNAs in exosomes as compared to donor platelets has been found. These results suggest that exosomes consistently contribute to PL activity and could represent an advantageous nanodelivery system for cell-free regeneration therapies.

  7. Exosome Proteome of U-87MG Glioblastoma Cells

    Directory of Open Access Journals (Sweden)

    Sohyun Chun

    2016-12-01

    Full Text Available Exosomes are small membrane vesicles between 30 and 100 nm in diameter secreted by many cell types, and are associated with a wide range of physiological and/or pathological processes. Exosomes containing proteins, lipids, mRNA, and microRNA contribute to cell-to-cell communication and cell-to-environment regulation, however, their biological functions are not yet fully understood. In this report, exosomes in the glioblastoma cell line, U-87MG, were isolated and the proteome was investigated. In addition, exosome proteome changes in U-87MG cells exposed to a low temperature were investigated to elucidate whether the exosome proteome could respond to an external stimulus. Cell culture medium was collected, and exosomes were isolated by continuous centrifugation eliminating cell debris, nucleic acids, and other particles. The morphology of exosomes was observed by cryo-tunneling electron microscopy. According to 2-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight mass spectrometry, certain proteins including collagen type VI alpha 1, putative RNA-binding protein 15B chain A, substrate induced remodeling of the active site regulates HTRA1, coatomer protein complex-subunit beta 2, myosin-heavy chain 1, and keratin-type I cytoskeletal 9 showed differences between the control proteome and the low temperature-exposed proteome.

  8. Exosomes from uninfected cells activate transcription of latent HIV-1.

    Science.gov (United States)

    Barclay, Robert A; Schwab, Angela; DeMarino, Catherine; Akpamagbo, Yao; Lepene, Benjamin; Kassaye, Seble; Iordanskiy, Sergey; Kashanchi, Fatah

    2017-07-14

    HIV-1 infection causes AIDS, infecting millions worldwide. The virus can persist in a state of chronic infection due to its ability to become latent. We have previously shown a link between HIV-1 infection and exosome production. Specifically, we have reported that exosomes transport viral proteins and RNA from infected cells to neighboring uninfected cells. These viral products could then elicit an innate immune response, leading to activation of the Toll-like receptor and NF-κB pathways. In this study, we asked whether exosomes from uninfected cells could activate latent HIV-1 in infected cells. We observed that irrespective of combination antiretroviral therapy, both short- and long-length viral transcripts were increased in wild-type HIV-1-infected cells exposed to purified exosomes from uninfected cells. A search for a possible mechanism for this finding revealed that the exosomes increase RNA polymerase II loading onto the HIV-1 promoter in the infected cells. These viral transcripts, which include trans-activation response (TAR) RNA and a novel RNA that we termed TAR-gag, can then be packaged into exosomes and potentially be exported to neighboring uninfected cells, leading to increased cellular activation. To better decipher the exosome release pathways involved, we used siRNA to suppress expression of ESCRT (endosomal sorting complex required for transport) proteins and found that ESCRT II and IV significantly control exosome release. Collectively, these results imply that exosomes from uninfected cells activate latent HIV-1 in infected cells and that true transcriptional latency may not be possible in vivo, especially in the presence of combination antiretroviral therapy.

  9. Exosomes as a liquid biopsy for lung cancer.

    Science.gov (United States)

    Cui, Shaohua; Cheng, Zhuoan; Qin, Wenxin; Jiang, Liyan

    2018-02-01

    In lung cancer and other malignancies, the so-called "liquid biopsy" is quickly moving into clinical practice. Its full potential has not yet been fully identified, but the "liquid biopsy" is no longer a promise but has become a reality that allows for better treatment selection and monitoring of lung cancer. This emerging field has significant potential to make up for the limitations of the traditional tissue-derived biomaterials. Exosomes are spherical nano-sized vesicles with a diameter of 40-100 nm and a density of 1.13-1.19 g/ml. In both physiological and pathological conditions, exosomes can be released by different cell types, including immune cells, stem cells and tumor cells. These small molecules may serve as promising biomarkers in lung cancer "liquid biopsy" as they can be easily obtained from most body fluids. In addition, the lipid bilayer of exosomes allows for stable cargoes which are relatively hard to degrade. Furthermore, the composition of exosomes reflects that of their parental cells, suggesting that exosomes are potential surrogates of the original cells and, therefore, are useful for understanding cell biology. Previous studies have demonstrated that exosomes play important roles in cell-to-cell communication. Moreover, tumor-derived exosomes are evolved in tumor-specific biological process, including tumor proliferation and progression. Recently, a growing number of studies has focused on exosomal cargo and their use in lung cancer genesis and progression. In addition, their utility as lung cancer diagnostic, prognostic and predictive biomarkers have also been studied. The current review primarily summaries lung cancer-related exosomal biomarkers that have recently been identified and discusses their potential in clinical practice. Copyright © 2017 Elsevier B.V. All rights reserved.

  10. Perturbations in the Urinary Exosome in Transplant Rejection

    Directory of Open Access Journals (Sweden)

    Tara eSigdel

    2015-01-01

    Full Text Available Urine exosomes are small vesicles exocytosed into the urine by all renal epithelial cell types under normal physiologic and disease states. Urine exosomal proteins may mirror disease specific proteome perturbations in kidney injury. Analysis methodologies for the exosomal fraction of the urinary proteome were developed for comparing the urinary exosomal fraction versus unfractionated proteome for biomarker discovery. Urine exosomes were isolated by centrifugal filtration of urine samples collected from kidney transplant patients with and without acute rejection, which were biopsy matched. The proteomes of unfractionated whole urine (Uw and urine exosomes (Ue underwent mass spectroscopy-based quantitative proteonomics analysis. The proteome data were analyzed for significant differential protein abundances in acute rejection (AR. A total of 1018 proteins were identified in Uw and 349 proteins in Ue. 279 overlapped between the two urinary compartments and 70 proteins were unique to the Ue compartment. Of 349 exosomal proteins identified from transplant patients,220 had not been previously identified in the normal Ue fraction. 11 Ue proteins, functionally involved in an inflammatory and stress response, were more abundant in urine samples from patients with acute rejection, 3 of which are exclusive to the Ue fraction. Ue AR-specific biomarkers(8 were also detected in Uw, but since they were observed at significantly lower abundances in Uw, they were not significant for AR in Uw. A rapid urinary exosome isolation method and quantitative measurement of enriched Ue proteins was applied. Perturbed proteins in the exosomal compartment of urine collected from kidney transplant patients were specific to inflammatory responses, and were not observed in the Ue fraction from normal healthy subjects. Ue specific protein alterations in renal disease provide potential mechanistic insights and offer a unique panel of sensitive biomarkers for monitoring AR.

  11. Syndecan-syntenin-ALIX regulates the biogenesis of exosomes.

    Science.gov (United States)

    Baietti, Maria Francesca; Zhang, Zhe; Mortier, Eva; Melchior, Aurélie; Degeest, Gisèle; Geeraerts, Annelies; Ivarsson, Ylva; Depoortere, Fabienne; Coomans, Christien; Vermeiren, Elke; Zimmermann, Pascale; David, Guido

    2012-06-03

    The biogenesis of exosomes, small secreted vesicles involved in signalling processes, remains incompletely understood. Here, we report evidence that the syndecan heparan sulphate proteoglycans and their cytoplasmic adaptor syntenin control the formation of exosomes. Syntenin interacts directly with ALIX through LYPX(n)L motifs, similarly to retroviral proteins, and supports the intraluminal budding of endosomal membranes. Syntenin exosomes depend on the availability of heparan sulphate, syndecans, ALIX and ESCRTs, and impact on the trafficking and confinement of FGF signals. This study identifies a key role for syndecan-syntenin-ALIX in membrane transport and signalling processes.

  12. Research Progress of Exosomes in Lung Cancer Diagnosis and Treatment

    Directory of Open Access Journals (Sweden)

    Hongbo ZOU

    2016-11-01

    Full Text Available As the leading cause of morbidity and cancer related-death worldwide, lung cancer has a serious threat to human health. Exosomes are nanoscale lipid membrane vesicles derived from multivesicles, which containing active biomolecules including proteins, lipids, nucleic acids and etc. Exosomes play important roles in lung cancer initiation and progression by promoting the formation of tumor microenvironment, enhancing tumor invasive and metastasis capability, leading to immunosuppression and resistance to chemoradiotherapy, and also have the application value in early diagnosis and treatment. This review summarizes the research progress of exosomes in tumor initiation and progression, and its roles in diagnosis and treatment of lung cancer.

  13. Interaction profiling identifies the human nuclear exosome targeting complex

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Christensen, Marianne Spangsberg; Kristiansen, Maiken Søndergaard

    2011-01-01

    The RNA exosome is a conserved degradation machinery, which obtains full activity only when associated with cofactors. The most prominent activator of the yeast nuclear exosome is the RNA helicase Mtr4p, acting in the context of the Trf4p/Air2p/Mtr4p polyadenylation (TRAMP) complex. The existence...... from nucleoli, and consistently NEXT is specifically required for the exosomal degradation of promoter upstream transcripts (PROMPTs). We also detect putative homolog TRAMP subunits hTRF4-2 (Trf4p) and ZCCHC7 (Air2p) in hRRP6 and hMTR4 precipitates. However, at least ZCCHC7 function is restricted...

  14. Angiogenic Mechanisms of Human CD34+ Stem Cell Exosomes in the Repair of Ischemic Hindlimb.

    Science.gov (United States)

    Mathiyalagan, Prabhu; Liang, Yaxuan; Kim, David; Misener, Sol; Thorne, Tina; Kamide, Christine E; Klyachko, Ekaterina; Losordo, Douglas W; Hajjar, Roger J; Sahoo, Susmita

    2017-04-28

    Paracrine secretions seem to mediate therapeutic effects of human CD34+ stem cells locally transplanted in patients with myocardial and critical limb ischemia and in animal models. Earlier, we had discovered that paracrine secretion from human CD34+ cells contains proangiogenic, membrane-bound nanovesicles called exosomes (CD34Exo). Here, we investigated the mechanisms of CD34Exo-mediated ischemic tissue repair and therapeutic angiogenesis by studying their miRNA content and uptake. When injected into mouse ischemic hindlimb tissue, CD34Exo, but not the CD34Exo-depleted conditioned media, mimicked the beneficial activity of their parent cells by improving ischemic limb perfusion, capillary density, motor function, and their amputation. CD34Exo were found to be enriched with proangiogenic miRNAs such as miR-126-3p. Knocking down miR-126-3p from CD34Exo abolished their angiogenic activity and beneficial function both in vitro and in vivo. Interestingly, injection of CD34Exo increased miR-126-3p levels in mouse ischemic limb but did not affect the endogenous synthesis of miR-126-3p, suggesting a direct transfer of stable and functional exosomal miR-126-3p. miR-126-3p enhanced angiogenesis by suppressing the expression of its known target, SPRED1, simultaneously modulating the expression of genes involved in angiogenic pathways such as VEGF (vascular endothelial growth factor), ANG1 (angiopoietin 1), ANG2 (angiopoietin 2), MMP9 (matrix metallopeptidase 9), TSP1 (thrombospondin 1), etc. Interestingly, CD34Exo, when treated to ischemic hindlimbs, were most efficiently internalized by endothelial cells relative to smooth muscle cells and fibroblasts, demonstrating a direct role of stem cell-derived exosomes on mouse endothelium at the cellular level. Collectively, our results have demonstrated a novel mechanism by which cell-free CD34Exo mediates ischemic tissue repair via beneficial angiogenesis. Exosome-shuttled proangiogenic miRNAs may signify amplification of stem

  15. Heparanase activates the syndecan-syntenin-ALIX exosome pathway

    National Research Council Canada - National Science Library

    Bart Roucourt Sofie Meeussen Jie Bao Pascale Zimmermann Guido David

    2015-01-01

    ... for transport accessory component ALIX. Here we investigated the role of heparanase, the only mammalian enzyme able to cleave heparan sulfate internally, in the syndecan-synten- in-ALIX exosome biogenesis pathway...

  16. Functional delivery of viral miRNAs via exosomes

    National Research Council Canada - National Science Library

    D. Michiel Pegtel; Katherine Cosmopoulos; David A. Thorley-Lawson; Monique A. J. van Eijndhoven; Erik S. Hopmans; Jelle L. Lindenberg; Tanja D. de Gruijl; Thomas Würdinger; Jaap M. Middeldorp; Elliott Kieff

    2010-01-01

    .... Interestingly, miRNAs are secreted actively through small vesicles called "exosomes" that protect them from degradation by RNases, suggesting that these miRNAs may function outside the cell in which they were produced...

  17. Exosome-bound WD repeat protein Monad inhibits breast cancer cell invasion by degrading amphiregulin mRNA.

    Directory of Open Access Journals (Sweden)

    Makio Saeki

    Full Text Available Increased stabilization of mRNA coding for key cancer genes can contribute to invasiveness. This is achieved by down-regulation of exosome cofactors, which bind to 3'-UTR in cancer-related genes. Here, we identified amphiregulin, an EGFR ligand, as a target of WD repeat protein Monad, a component of R2TP/prefoldin-like complex, in MDA-MB-231 breast cancer cells. Monad specifically interacted with both the 3'-UTR of amphiregulin mRNA and the RNA degrading exosome, and enhanced decay of amphiregulin transcripts. Knockdown of Monad increased invasion and this effect was abolished with anti-amphiregulin neutralizing antibody. These results suggest that Monad could prevent amphiregulin-mediated invasion by degrading amphiregulin mRNA.

  18. Mediatization

    DEFF Research Database (Denmark)

    Hjarvard, Stig

    2017-01-01

    Mediatization research shares media effects studies' ambition of answering the difficult questions with regard to whether and how media matter and influence contemporary culture and society. The two approaches nevertheless differ fundamentally in that mediatization research seeks answers...... to these general questions by distinguishing between two concepts: mediation and mediatization. The media effects tradition generally considers the effects of the media to be a result of individuals being exposed to media content, i.e. effects are seen as an outcome of mediated communication. Mediatization....... From the perspective of mediatization research, the most important effect of the media stems from their embeddedness in culture and society....

  19. NVL2, a nucleolar AAA-ATPase, is associated with the nuclear exosome and is involved in pre-rRNA processing

    Energy Technology Data Exchange (ETDEWEB)

    Yoshikatsu, Yuki [Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima 770-8506 (Japan); Ishida, Yo-ichi; Sudo, Haruka [Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan); Yuasa, Keizo; Tsuji, Akihiko [Department of Life Systems, Institute of Technology and Science, The University of Tokushima Graduate School, Tokushima 770-8506 (Japan); Nagahama, Masami, E-mail: nagahama@my-pharm.ac.jp [Department of Molecular and Cellular Biochemistry, Meiji Pharmaceutical University, Kiyose, Tokyo 204-8588 (Japan)

    2015-08-28

    Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex. - Highlights: • ATPase-deficient mutants of NVL2 have decreased pre-rRNA processing. • NVL2 associates with the nuclear exosome through interactions with MTR4 and RRP6. • MPP6 stabilizes MTR4-RRP6 interaction and allows NVL2 to interact with the complex.

  20. Effects of storage temperature on airway exosome integrity for diagnostic and functional analyses.

    Science.gov (United States)

    Maroto, Rosario; Zhao, Yingxin; Jamaluddin, Mohammad; Popov, Vsevolod L; Wang, Hongwang; Kalubowilage, Madumali; Zhang, Yueqing; Luisi, Jonathan; Sun, Hong; Culbertson, Christopher T; Bossmann, Stefan H; Motamedi, Massoud; Brasier, Allan R

    2017-01-01

    Background: Extracellular vesicles contain biological molecules specified by cell-type of origin and modified by microenvironmental changes. To conduct reproducible studies on exosome content and function, storage conditions need to have minimal impact on airway exosome integrity. Aim: We compared surface properties and protein content of airway exosomes that had been freshly isolated vs. those that had been treated with cold storage or freezing. Methods: Mouse bronchoalveolar lavage fluid (BALF) exosomes purified by differential ultracentrifugation were analysed immediately or stored at +4°C or -80°C. Exosomal structure was assessed by dynamic light scattering (DLS), transmission electron microscopy (TEM) and charge density (zeta potential, ζ). Exosomal protein content, including leaking/dissociating proteins, were identified by label-free LC-MS/MS. Results: Freshly isolated BALF exosomes exhibited a mean diameter of 95 nm and characteristic morphology. Storage had significant impact on BALF exosome size and content. Compared to fresh, exosomes stored at +4°C had a 10% increase in diameter, redistribution to polydisperse aggregates and reduced ζ. Storage at -80°C produced an even greater effect, resulting in a 25% increase in diameter, significantly reducing the ζ, resulting in multilamellar structure formation. In fresh exosomes, we identified 1140 high-confidence proteins enriched in 19 genome ontology biological processes. After storage at room temperature, 848 proteins were identified. In preparations stored at +4°C, 224 proteins appeared in the supernatant fraction compared to the wash fractions from freshly prepared exosomes; these proteins represent exosome leakage or dissociation of loosely bound "peri-exosomal" proteins. In preparations stored at -80°C, 194 proteins appeared in the supernatant fraction, suggesting that distinct protein groups leak from exosomes at different storage temperatures. Conclusions: Storage destabilizes the surface

  1. Exosomes derived from endometriotic stromal cells have enhanced angiogenic effects in vitro.

    Science.gov (United States)

    Harp, Djana; Driss, Adel; Mehrabi, Sharifeh; Chowdhury, Indrajit; Xu, Wei; Liu, Dong; Garcia-Barrio, Minerva; Taylor, Robert N; Gold, Bert; Jefferson, Samantha; Sidell, Neil; Thompson, Winston

    2016-07-01

    Our objective has been to establish a pro-angiogenic role for exosomes in endometriosis and to determine whether a differential expression profile of cellular and exosomal microRNAs (miRNAs) exists in endometriosis. We performed an in vitro study of human primary endometrial stromal cells (ESCs) and human umbilical vein endothelial cells (HUVECs). We isolated and characterized exosomes from ESCs from five endometriosis patients and five phase-matched controls. Exosomes were characterized by transmission electron microscopy and NanoSight technology. MiRNA was assessed by deep sequencing and reverse transcription with quantitative polymerase chain reaction. Exosome uptake studies were achieved by means of confocal microscopy. The pro-angiogenic experiments were executed by treating HUVECs with ESC-derived exosomes. We observed differential profiles of exosomal miRNA expression between exosomes derived from endometriosis lesion cells and diseased eutopic stromal cells compared with exosomes derived from control ESCs. We also demonstrated autocrine cellular uptake of exosomes and paracrine functional angiogenic effects of exosomes on HUVECs. The results of this study support the hypothesis that exosomes derived from ESCs play autocrine/paracrine roles in the development of endometriosis, potentially modulating angiogenesis. The broader clinical implications are that Sampson's theory of retrograde menstruation possibly encompasses the finding that exosomes work as intercellular communication modulators in endometriosis.

  2. Tumor-derived exosomes in cancer progression and treatment failure

    Science.gov (United States)

    Shen, Bo; Feng, Jifeng

    2015-01-01

    Exosomes have diameter within the range of 30-100nm and spherical to cup-shaped nanoparticles with specific surface molecular characteristics, such as CD9 and CD63. These vesicles are present in nearly all human body fluids, including blood plasma/serum, saliva, breast milk, cerebrospinal fluid, urine, semen, and particularly enriched in tumor microenvironment. Exosomes contain multiple proteins, DNA, mRNA, miRNA, long non-coding RNA, and even genetic materials of viruses/prions. These materials are biochemically and functionally distinct and can be transferred to a recipient cell where they regulate protein expression and signaling pathways. Recently, exosomes are demonstrated to have a close relationship with tumor development and metastasis. Exosomes influence therapeutic effect in cancer patients. In this review, we describe the biogenesis, composition, and function of exosomes. The mechanism on how tumor-derived exosomes contribute to cancer progression and clinical treatment failure is also described, with special focus on their potential applications in cancer therapy. PMID:26452221

  3. Perspective Insights of Exosomes in Neurodegenerative Diseases: A Critical Appraisal

    Directory of Open Access Journals (Sweden)

    Arif Tasleem Jan

    2017-09-01

    Full Text Available Exosomes are small membranous entities of endocytic origin. Their production by a wide variety of cells in eukaryotes implicates their roles in the execution of essential processes, especially cellular communication. Exosomes are secreted under both physiological and pathophysiological conditions, and their actions on neighboring and distant cells lead to the modulations of cellular behaviors. They also assist in the delivery of disease causing entities, such as prions, α-syn, and tau, and thus, facilitate spread to non-effected regions and accelerate the progressions of neurodegenerative diseases. The characterization of exosomes, provides information on aberrant processes, and thus, exosome analysis has many clinical applications. Because they are associated with the transport of different cellular entities across the blood-brain barrier (BBB, exosomes might be useful for delivering drugs and other therapeutic molecules to brain. Herein, we review roles played by exosomes in different neurodegenerative diseases, and the possibilities of using them as diagnostic biomarkers of disease progression, drug delivery vehicles and in gene therapy.

  4. Perspective Insights of Exosomes in Neurodegenerative Diseases: A Critical Appraisal

    Science.gov (United States)

    Jan, Arif Tasleem; Malik, Mudasir A.; Rahman, Safikur; Yeo, Hye R.; Lee, Eun J.; Abdullah, Tasduq S.; Choi, Inho

    2017-01-01

    Exosomes are small membranous entities of endocytic origin. Their production by a wide variety of cells in eukaryotes implicates their roles in the execution of essential processes, especially cellular communication. Exosomes are secreted under both physiological and pathophysiological conditions, and their actions on neighboring and distant cells lead to the modulations of cellular behaviors. They also assist in the delivery of disease causing entities, such as prions, α-syn, and tau, and thus, facilitate spread to non-effected regions and accelerate the progressions of neurodegenerative diseases. The characterization of exosomes, provides information on aberrant processes, and thus, exosome analysis has many clinical applications. Because they are associated with the transport of different cellular entities across the blood-brain barrier (BBB), exosomes might be useful for delivering drugs and other therapeutic molecules to brain. Herein, we review roles played by exosomes in different neurodegenerative diseases, and the possibilities of using them as diagnostic biomarkers of disease progression, drug delivery vehicles and in gene therapy. PMID:29033828

  5. An innovative method for exosome quantification and size measurement.

    Science.gov (United States)

    Mehdiani, Arash; Maier, Anatol; Pinto, Antonio; Barth, Mareike; Akhyari, Payam; Lichtenberg, Artur

    2015-01-17

    Although the biological importance of exosomes has recently gained an increasing amount of scientific and clinical attention, much is still unknown about their complex pathways, their bioavailability and their diverse functions in health and disease. Current work focuses on the presence and the behavior of exosomes (in vitro as well as in vivo) in the context of different human disorders, especially in the fields of oncology, gynecology and cardiology. Unfortunately, neither a consensus regarding a gold standard for exosome isolation exists, nor is there an agreement on such a method for their quantitative analysis. As there are many methods for the purification of exosomes and also many possibilities for their quantitative and qualitative analysis, it is difficult to determine a combination of methods for the ideal approach. Here, we demonstrate nanoparticle tracking analysis (NTA), a semi-automated method for the characterization of exosomes after isolation from human plasma by ultracentrifugation. The presented results show that this approach for isolation, as well as the determination of the average number and size of exosomes, delivers reproducible and valid data, as confirmed by other methods, such as scanning electron microscopy (SEM).

  6. Exosome Cofactors Connect Transcription Termination to RNA Processing by Guiding Terminated Transcripts to the Appropriate Exonuclease within the Nuclear Exosome.

    Science.gov (United States)

    Kim, Kyumin; Heo, Dong-Hyuk; Kim, Iktae; Suh, Jeong-Yong; Kim, Minkyu

    2016-06-17

    The yeast Nrd1 interacts with the C-terminal domain (CTD) of RNA polymerase II (RNApII) through its CTD-interacting domain (CID) and also associates with the nuclear exosome, thereby acting as both a transcription termination and RNA processing factor. Previously, we found that the Nrd1 CID is required to recruit the nuclear exosome to the Nrd1 complex, but it was not clear which exosome subunits were contacted. Here, we show that two nuclear exosome cofactors, Mpp6 and Trf4, directly and competitively interact with the Nrd1 CID and differentially regulate the association of Nrd1 with two catalytic subunits of the exosome. Importantly, Mpp6 promotes the processing of Nrd1-terminated transcripts preferentially by Dis3, whereas Trf4 leads to Rrp6-dependent processing. This suggests that Mpp6 and Trf4 may play a role in choosing a particular RNA processing route for Nrd1-terminated transcripts within the exosome by guiding the transcripts to the appropriate exonuclease. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  7. EXOSOMES: CAN DOCTORS STILL IGNORE THEIR EXISTENCE?

    Directory of Open Access Journals (Sweden)

    Celeste Caruso Bavisotto

    2013-04-01

    Full Text Available With this invited commentary we want to draw the attention of young medical doctors, the main readers of this journal, towards the existence and importance of a group of nanovesicles released by human cells: the exosomes. These vesicles are incontinently secreted as a mean of cell-to-cell communication. They are involved in a number of physiologic processes as well as in the pathogenesis of, virtually, all human diseases. They can be isolated from all biological fluids, like blood, urine, sweat, sperm, crevicular fluid, bile, etc., and their composition in terms of proteins, RNA and lipids is different in pathology that in physiologic conditions. It is therefore possible to predict that they will become an important diagnostic and therapeutic tool in medicine.

  8. Exosomes Derived from Squamous Head and Neck Cancer Promote Cell Survival after Ionizing Radiation

    Science.gov (United States)

    Mutschelknaus, Lisa; Peters, Carsten; Winkler, Klaudia; Yentrapalli, Ramesh; Heider, Theresa; Atkinson, Michael John; Moertl, Simone

    2016-01-01

    Exosomes are nanometer-sized extracellular vesicles that are believed to function as intercellular communicators. Here, we report that exosomes are able to modify the radiation response of the head and neck cancer cell lines BHY and FaDu. Exosomes were isolated from the conditioned medium of irradiated as well as non-irradiated head and neck cancer cells by serial centrifugation. Quantification using NanoSight technology indicated an increased exosome release from irradiated compared to non-irradiated cells 24 hours after treatment. To test whether the released exosomes influence the radiation response of other cells the exosomes were transferred to non-irradiated and irradiated recipient cells. We found an enhanced uptake of exosomes isolated from both irradiated and non-irradiated cells by irradiated recipient cells compared to non-irradiated recipient cells. Functional analyses by exosome transfer indicated that all exosomes (from non-irradiated and irradiated donor cells) increase the proliferation of non-irradiated recipient cells and the survival of irradiated recipient cells. The survival-promoting effects are more pronounced when exosomes isolated from irradiated compared to non-irradiated donor cells are transferred. A possible mechanism for the increased survival after irradiation could be the increase in DNA double-strand break repair monitored at 6, 8 and 10 h after the transfer of exosomes isolated from irradiated cells. This is abrogated by the destabilization of the exosomes. Our results demonstrate that radiation influences both the abundance and action of exosomes on recipient cells. Exosomes transmit prosurvival effects by promoting the proliferation and radioresistance of head and neck cancer cells. Taken together, this study indicates a functional role of exosomes in the response of tumor cells to radiation exposure within a therapeutic dose range and encourages that exosomes are useful objects of study for a better understanding of tumor

  9. Exosomes as potent regulators of HCC malignancy and potential bio-tools in clinical application

    OpenAIRE

    Qu, Zhen; Jiang, Chunping; Wu, Junhua; Ding, Yitao

    2015-01-01

    Exosomes are small membranous vesicles about 30~100 nm in diameter and formed from inward budding of the limiting membrane of multi-vesicular bodies (MVB). Exosomes are secreted by most cell types (including hepatocellular carcinoma cells) into the extracellular environment and can be isolated from various body fluids. Exosomes have broad biological function through delivering contained molecules to the target cells. Although limited studies on hepatocellular carcinoma (HCC) exosomes, increas...

  10. Isolation and characterization of exosomes from cell culture supernatants and biological fluids

    OpenAIRE

    Théry, Clotilde; Amigorena, Sebastian; Raposo, Graça; Clayton, Aled

    2006-01-01

    Exosomes are small membrane vesicles found in cell culture supernatants and in different biological fluids. Exosomes form in a particular population of endosomes, called multivesicular bodies (MVBs), by inward budding into the lumen of the compartment. Upon fusion of MVBs with the plasma membrane, these internal vesicles are secreted. Exosomes possess a defined set of membrane and cytosolic proteins. The physiological function of exosomes is still a matter of debate, but increasing results in...

  11. Purified horse milk exosomes contain an unpredictable small number of major proteins

    Directory of Open Access Journals (Sweden)

    Sergey E. Sedykh

    2017-06-01

    The article describes the morphology and the protein content of major horse milk exosomes for the first time. Our results on the decrease of major protein number identified in exosomal preparations after gel filtration may be important to the studies of biological functions of pure exosomes.

  12. Announcing , the Official Journal of the American Society for Exosomes and Microvesicles

    Directory of Open Access Journals (Sweden)

    Stephen J. Gould

    2013-01-01

    Full Text Available This editorial article introduces the new scientific journal Exosomes and Microvesicles (EXMV , the official journal of the American Society for Exosomes and Microvesicles (ASEMV, and describes its editorial line and mission in relation to the role of the Society, the state of the art of the study of exosomes and microvesicles, and the overall approach of the publication.

  13. Dendritic cell-derived exosomes elicit tumor regression in autochthonous hepatocellular carcinoma mouse models.

    Science.gov (United States)

    Lu, Zhen; Zuo, Bingfeng; Jing, Renwei; Gao, Xianjun; Rao, Quan; Liu, Zhili; Qi, Han; Guo, Hongxing; Yin, HaiFang

    2017-10-01

    Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC), a life-threatening malignancy with limited treatment options in the clinic that responds poorly to immunotherapy, remains to be investigated. Exosomes derived from α-fetoprotein (AFP)-expressing DCs (DEXAFP) were investigated in three different HCC mouse models systemically. Tumor growth and microenvironment were monitored. DEXAFP elicited strong antigen-specific immune responses and resulted in significant tumor growth retardation and prolonged survival rates in mice with ectopic, orthotopic and carcinogen-induced HCC tumors that displayed antigenic and pathological heterogeneity. The tumor microenvironment was improved in DEXAFP-treated HCC mice, demonstrated by significantly more γ-interferon (IFN-γ)-expressing CD8+ T lymphocytes, elevated levels of IFN-γ and interleukin-2, and fewer CD25+Foxp3+ regulatory T (Treg) cells and decreased levels of interleukin-10 and transforming growth factor-β in tumor sites. Lack of efficacy in athymic nude mice and CD8+ T cell-depleted mice showed that T cells contribute to DEXAFP-mediated antitumor function. Dynamic examination of the antitumor efficacy and the immune microenvironment in DEXAFP-treated orthotopic HCC mice at different time-points revealed a positive correlation between tumor suppression and immune microenvironment. Our findings provide evidence that AFP-enriched DEXs can trigger potent antigen-specific antitumor immune responses and reshape the tumor microenvironment in HCC mice and thus provide a cell-free vaccine option for HCC immunotherapy. Lay summary: Dendritic cell (DC)-derived exosomes (DEXs) form a new class of vaccines for cancer immunotherapy. However, their potency in hepatocellular carcinoma (HCC) remains unknown. Here, we investigated exosomes from HCC antigen-expressing DCs in three different HCC mouse models and proved their feasibility

  14. Altered expression of CD63 and exosomes in scleroderma dermal fibroblasts.

    Science.gov (United States)

    Nakamura, Kayo; Jinnin, Masatoshi; Harada, Miho; Kudo, Hideo; Nakayama, Wakana; Inoue, Kuniko; Ogata, Aki; Kajihara, Ikko; Fukushima, Satoshi; Ihn, Hironobu

    2016-10-01

    Exosomes are small vesicles shed from various cells. They contain proteins, lipids, and nucleic acids, and are regarded as a tool of cell-cell communication. To reveal the putative role of exosomes in systemic sclerosis (SSc), and to elucidate the effect of exosomes on wound healing. The expression of common markers for exosomes (CD63, CD9, and CD81) and type I collagen were examined with real-time PCR, immunohistochemical analysis, ELISA, immunoblotting, and flow cytometry. The effect of serum-derived exosomes on wound healing was tested on full-thickness wounds in the mid-dorsal skin of BALB/c mice. The expression levels of CD63 as well as CD9 and CD81 tended to be increased in SSc dermal fibroblasts compared to normal fibroblasts. Increased exosomes in a cultured media of SSc fibroblasts stimulated the expression levels of type I collagen in normal fibroblasts. As the mechanism, collagen-related microRNA levels in SSc fibroblast-derived exosomes were dysregulated, indicating that both the amount and the content of exosomes were altered in SSc. On the other hand, SSc sera showed significantly decreased exosome levels compared to normal sera. The frequencies of vascular involvements, including skin ulcers or pitting scars, were significantly increased in patients with decreased serum exosome levels. The healing of mice wounds was accelerated by treatment with serum-derived exosomes. Vascular abnormalities in SSc may account for the decreased serum exosome levels by the disturbed transfer of exosomes from the skin tissue to the blood stream. Our study suggests the possibility that SSc patients with vascular involvements have decreased serum exosome levels, which causes the delay of wound healing due to down-regulation of collagen, resulting in higher susceptibility to pitting scars and/or ulcers. Exosome research will lead to a detailed understanding of SSc pathogenesis and new therapeutic approaches. Copyright © 2016 Japanese Society for Investigative Dermatology

  15. Human vascular endothelial cells transport foreign exosomes from cow's milk by endocytosis.

    Science.gov (United States)

    Kusuma, Rio Jati; Manca, Sonia; Friemel, Taylor; Sukreet, Sonal; Nguyen, Christopher; Zempleni, Janos

    2016-05-15

    Encapsulation of microRNAs in exosomes confers protection against degradation and a vehicle for shuttling of microRNAs between cells and tissues, and cellular uptake by endocytosis. Exosomes can be found in foods including milk. Humans absorb cow's milk exosomes and deliver the microRNA cargo to peripheral tissues, consistent with gene regulation by dietary nucleic acids across species boundaries. Here, we tested the hypothesis that human vascular endothelial cells transport milk exosomes by endocytosis, constituting a step crucial for the delivery of dietary exosomes and their cargo to peripheral tissues. We tested this hypothesis by using human umbilical vein endothelial cells and fluorophore-labeled exosomes isolated from cow's milk. Exosome uptake followed Michaelis-Menten kinetics (Vmax = 0.057 ± 0.004 ng exosome protein × 40,000 cells/h; Km = 17.97 ± 3.84 μg exosomal protein/200 μl media) and decreased by 80% when the incubation temperature was lowered from 37°C to 4°C. When exosome surface proteins were removed by treatment with proteinase K, or transport was measured in the presence of the carbohydrate competitor d-galactose or measured in the presence of excess unlabeled exosomes, transport rates decreased by 45% to 80% compared with controls. Treatment with an inhibitor of endocytosis, cytochalasin D, caused a 50% decrease in transport. When fluorophore-labeled exosomes were administered retro-orbitally, exosomes accumulated in liver, spleen, and lungs in mice. We conclude that human vascular endothelial cells transport bovine exosomes by endocytosis and propose that this is an important step in the delivery of dietary exosomes and their cargo to peripheral tissues. Copyright © 2016 the American Physiological Society.

  16. Distinct Dasatinib-Induced Mechanisms of Apoptotic Response and Exosome Release in Imatinib-Resistant Human Chronic Myeloid Leukemia Cells

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    Juan Liu

    2016-04-01

    Full Text Available Although dasatinib is effective in most imatinib mesylate (IMT-resistant chronic myeloid leukemia (CML patients, the underlying mechanism of its effectiveness in eliminating imatinib-resistant cells is only partially understood. This study investigated the effects of dasatinib on signaling mechanisms driving-resistance in imatinib-resistant CML cell line K562 (K562RIMT. Compared with K562 control cells, exsomal release, the phosphoinositide 3-kinase (PI3K/protein kinase B (Akt/ mammalian target of rapamycin (mTOR signaling and autophagic activity were increased significantly in K562RIMT cells and mTOR-independent beclin-1/Vps34 signaling was shown to be involved in exosomal release in these cells. We found that Notch1 activation-mediated reduction of phosphatase and tensin homolog (PTEN was responsible for the increased Akt/mTOR activities in K562RIMT cells and treatment with Notch1 γ-secretase inhibitor prevented activation of Akt/mTOR. In addition, suppression of mTOR activity by rapamycin decreased the level of activity of p70S6K, induced upregulation of p53 and caspase 3, and led to increase of apoptosis in K562RIMT cells. Inhibition of autophagy by spautin-1 or beclin-1 knockdown decreased exosomal release, but did not affect apoptosis in K562RIMT cells. In summary, in K562RIMT cells dasatinib promoted apoptosis through downregulation of Akt/mTOR activities, while preventing exosomal release and inhibiting autophagy by downregulating expression of beclin-1 and Vps34. Our findings reveal distinct dasatinib-induced mechanisms of apoptotic response and exosomal release in imatinib-resistant CML cells.

  17. Circulating exosomes as new biomarkers for brain disease and injury

    Science.gov (United States)

    Graner, Michael W.; Epple, Laura M.; Dusto, Nathaniel L.; Lencioni, Alex M.; Nega, Meheret; Herring, Matthew; Winston, Ben; Madsen, Helen; Bemis, Lynne T.; Anchordoquy, Thomas J.

    2013-05-01

    Brain diseases such as cancers, neurodegenerative disorders, or trauma are frequently diagnosed with imaging modalities and sometimes with intracranial biopsies. Treatment response is similarly monitored, along with clinical indications. While these technologies provide important windows into the disease state, they fail to provide us a detailed molecular portrait of the disease and of the changes taking place during therapy. Exosomes are virus-sized nanovesicles derived from the endosomal system and are released extracellularly from essentially all cell types. Exosomes contain intracellular entities (proteins, nucleic acids, metabolites), membrane proteins and lipids, and even extracellular proteins bound to them. Exosomes may be considered as mini-surrogates of their cells of origin, with some content common to all cells/exosomes, but some of the content would be cell-specific. These vesicles are found in all biofluids in humans, and are thus accessible to "liquid biopsy" with harvest of vesicles from such fluids. Current challenges are to identify disease-related markers or panels of markers to distinguish the disease state. Here we will show examples of brain tumor markers found in/on exosomes from cell culture and patient sera, and we will suggest that aspects of the biology of disease may have a relevant place in the search for biomarkers.

  18. Molecular lipidomics of exosomes released by PC-3 prostate cancer cells

    DEFF Research Database (Denmark)

    Llorente, A.; Skotland, T.; Sylvanne, T.

    2013-01-01

    The molecular lipid composition of exosomes is largely unknown. In this study, sophisticated shotgun and targeted molecular lipidomic assays were performed for in-depth analysis of the lipidomes of the metastatic prostate cancer cell line, PC-3, and their released exosomes. This study, based...... in the quantification of approximately 280 molecular lipid species, provides the most extensive lipid analysis of cells and exosomes to date. Interestingly, major differences were found in the lipid composition of exosomes compared to parent cells. Exosomes show a remarkable enrichment of distinct lipids, demonstrating...

  19. Exosomal proteins as prognostic biomarkers in non-small cell lung cancer

    DEFF Research Database (Denmark)

    Paulsen, Birgitte Sandfeld; Aggerholm-Pedersen, N; Bæk, R

    2016-01-01

    for optimal treatment. We here evaluate exosomes by protein phenotyping as a prognostic biomarker in NSCLC. METHODS: Exosomes from plasma of 276 NSCLC patients were phenotyped using the Extracellular Vesicle Array; 49 antibodies captured the proteins on the exosomes, and a cocktail of biotin......BACKGROUND: Use of exosomes as biomarkers in non-small cell lung cancer (NSCLC) is an intriguing approach in the liquid-biopsy era. Exosomes are nano-sized vesicles with membrane-bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection...

  20. Exosomes participate in the carcinogenesis and the malignant behavior of gastric cancer.

    Science.gov (United States)

    Yan, Yunmeng; Fu, Guangzhen; Ye, Yafei; Ming, Liang

    2017-05-01

    In order to summarize the role of exosomes in invasion and metastasis in gastric cancer (GC). Exosomes are vesicles of endocytic origin ranging from 30 to 100 nm in size; they are composed of a lipid bilayer and contain DNA, mRNA, miRNA, circular RNA and multiple proteins. Recently, increasing evidence shows that exosomes play a crucial role in the tumorigenesis of GC. In this review, we focus on the latest findings on GC exosomes, mainly summarizing their role in invasion and metastasis in GC. Then, exosomes? potential functions as novel diagnostic and therapeutic biomarkers for GC are briefly discussed. At last, we prospect the clinical application perspective of exosomes in GC. Exosomes play a vital role in gastric cancer carcinogenesis and metastasis.

  1. The Role of Exosomal VP40 in Ebola Virus Disease.

    Science.gov (United States)

    Pleet, Michelle L; DeMarino, Catherine; Lepene, Benjamin; Aman, M Javad; Kashanchi, Fatah

    2017-04-01

    Ebola virus (EBOV) can cause a devastating hemorrhagic disease, leading to death in a short period of time. After infection, the resulting EBOV disease results in high levels of circulating cytokines, endothelial dysfunction, coagulopathy, and bystander lymphocyte apoptosis in humans and nonhuman primates. The VP40 matrix protein of EBOV is essential for viral assembly and budding from the host cell. Recent data have shown that VP40 exists in the extracellular environment, including in exosomes, and exosomal VP40 can impact the viability of recipient immune cells, including myeloid and T cells, through the regulation of the RNAi and endosomal sorting complexes required for transport pathways. In this study, we discuss the latest findings of the impact of exosomal VP40 on immune cells in vitro and its potential implications for pathogenesis in vivo.

  2. Role of Exosomes in Placental Homeostasis and Pregnancy Disorders.

    Science.gov (United States)

    Salomon, C; Rice, G E

    2017-01-01

    The human placenta is a unique organ that performs the function of the majority of fetal organs across gestation. How the placenta communicates with maternal tissues to prepare them for pregnancy is not fully understood. Recently, it has been established that placental cells can communicate with maternal tissues to regulate their biological function via extracellular vesicles (EVs). EVs are subclassified into exosomes or microvesicles (MVs) according to their size, cell or tissue of origin, functions, and physical features. Exosomes are a specific type of EVs from an endocytic origin, while MVs are released via budding from the plasma membrane. With regards to pregnancy, the role of EVs has been described in several functions such as immune responses and maternal metabolic adaptation to gestation. Interestingly, EVs of placental origin can be detected in a variety of body fluids including urine and blood, and have been identified in the maternal circulation at as early as 6 weeks of gestation. Moreover, the number of exosomes across gestation is higher in complications of pregnancies such as preeclampsia and gestational diabetes mellitus compared to normal pregnancies. Circulating exosomes contains proteins and RNAs that are representative of the cell of origin, including surface and cytoplasmic protein, messenger RNA, and micro-RNAs. Finally, exosomes are capable of transferring their contents to other cells and regulating the biological function of the target cell. In this review, we will discuss the effect of the maternal microenvironment on secretion and content of placenta-derived EVs, and how this may lead to complications of pregnancies with a special emphasis on exosomes. © 2017 Elsevier Inc. All rights reserved.

  3. Suppression of endothelial cell migration by tumor associated macrophage-derived exosomes is reversed by epithelial ovarian cancer exosomal lncRNA.

    Science.gov (United States)

    Wu, Quanfeng; Wu, Xiaoli; Ying, Xiang; Zhu, Qinyi; Wang, Xinjing; Jiang, Lu; Chen, Xin; Wu, Yueqian; Wang, Xipeng

    2017-01-01

    To study the mechanism by which epithelial ovarian cancer (EOC)-derived exosomes restore the migration of endothelial cells that is suppressed by TAM-derived exosomes. Exosomes were isolated from TAMs in the ascites of patients with EOC. The effect of exosomes on the expression of endothelial cell miRNA was monitored by PCR. The miRNA mimics were transfected to explore their effects. Microarray data and literature searches were used to predict target genes and the impact of target gene pathways, and small interfering RNA was used to target these genes. We used migration assays to determine whether ovarian cancer cell-derived exosomes participate in the regulation of TAMs and endothelial cells. We used microarray data to identify the target lncRNA, and we constructed target lncRNA expression plasmids to validate targets by Western blotting. We separated TAMs from the ascites of patients with EOC and isolated exosomes from TAM supernatants. After co-culture with HUVECs, these exosomes were efficiently incorporated into HUVECs. The migration of HUVECs was suppressed significantly in the exosome group compared with blank controls (P derived exosomes targeted the miR-146b-5p/TRAF6/NF-κB/MMP2 pathway to suppress endothelial cell migration; this result was supported by PCR and Western blotting analyses. The expression of exosomal miR-146b-5p isolated from serum in the EOC group was significantly increased compared to healthy individuals. Finally, TAM-derived exosomes and EOC SKOV3-derived exosomes in combination stimulated HUVEC cells and overcame the inhibition of endothelial cell migration caused by TAM-derived exosomes. Two lncRNAs that were carried by SKOV3-derived exosomes were identified as NF-κB pathway-associated genes by Western blotting. TAM-derived exosomes can inhibit the migration of endothelial cells by targeting the miR-146b-5p/TRAF6/NF-kB/MMP2 pathway. However, EOC-derived exosomes can transfer lncRNAs to remotely reverse this effect of TAMs on

  4. High-resolution proteomic and lipidomic analysis of exosomes and microvesicles from different cell sources

    Directory of Open Access Journals (Sweden)

    Reka A. Haraszti

    2016-11-01

    Full Text Available Extracellular vesicles (EVs, including exosomes and microvesicles (MVs, are explored for use in diagnostics, therapeutics and drug delivery. However, little is known about the relationship of protein and lipid composition of EVs and their source cells. Here, we report high-resolution lipidomic and proteomic analyses of exosomes and MVs derived by differential ultracentrifugation from 3 different cell types: U87 glioblastoma cells, Huh7 hepatocellular carcinoma cells and human bone marrow-derived mesenchymal stem cells (MSCs. We identified 3,532 proteins and 1,961 lipid species in the screen. Exosomes differed from MVs in several different areas: (a The protein patterns of exosomes were more likely different from their cells of origin than were the protein patterns of MVs; (b The proteomes of U87 and Huh7 exosomes were similar to each other but different from the proteomes of MSC exosomes, whereas the lipidomes of Huh7 and MSC exosomes were similar to each other but different from the lipidomes of U87 exosomes; (c exosomes exhibited proteins of extracellular matrix, heparin-binding, receptors, immune response and cell adhesion functions, whereas MVs were enriched in endoplasmic reticulum, proteasome and mitochondrial proteins. Exosomes and MVs also differed in their types of lipid contents. Enrichment in glycolipids and free fatty acids characterized exosomes, whereas enrichment in ceramides and sphingomyelins characterized MVs. Furthermore, Huh7 and MSC exosomes were specifically enriched in cardiolipins; U87 exosomes were enriched in sphingomyelins. This study comprehensively analyses the protein and lipid composition of exosomes, MVs and source cells in 3 different cell types.

  5. The human core exosome interacts with differentially localized processive RNases

    DEFF Research Database (Denmark)

    Tomecki, Rafal; Kristiansen, Maiken Søndergaard; Lykke-Andersen, Søren

    2010-01-01

    The eukaryotic RNA exosome is a ribonucleolytic complex involved in RNA processing and turnover. It consists of a nine-subunit catalytically inert core that serves a structural function and participates in substrate recognition. Best defined in Saccharomyces cerevisiae, enzymatic activity comes......, identity and arrangements of its catalytic subunits in different vertebrates remain elusive. Here, we demonstrate the association of two different Dis3p homologs--hDIS3 and hDIS3L--with the human exosome core. Interestingly, these factors display markedly different intracellular localizations: hDIS3...

  6. Cisplatin-resistant lung cancer cell-derived exosomes increase cisplatin resistance of recipient cells in exosomal miR-100-5p-dependent manner.

    Science.gov (United States)

    Qin, Xiaobing; Yu, Shaorong; Zhou, Leilei; Shi, Meiqi; Hu, Yong; Xu, Xiaoyue; Shen, Bo; Liu, Siwen; Yan, Dali; Feng, Jifeng

    2017-01-01

    Exosomes derived from lung cancer cells confer cisplatin (DDP) resistance to other cancer cells. However, the underlying mechanism is still unknown. A549 resistance to DDP (A549/DDP) was established. Microarray was used to analyze microRNA (miRNA) expression profiles of A549 cells, A549/DDP cells, A549 exosomes, and A549/DDP exosomes. There was a strong correlation of miRNA profiles between exosomes and their maternal cells. A total of 11 miRNAs were significantly upregulated both in A549/DDP cells compared with A549 cells and in exosomes derived from A549/DDP cells in contrast to exosomes from A549 cells. A total of 31 downregulated miRNAs were also observed. miR-100-5p was the most prominent decreased miRNA in DDP-resistant exosomes compared with the corresponding sensitive ones. Downregulated miR-100-5p was proved to be involved in DDP resistance in A549 cells, and mammalian target of rapamycin (mTOR) expression was reverse regulated by miR-100-5p. Exosomes confer recipient cells' resistance to DDP in an exosomal miR-100-5p-dependent manner with mTOR as its potential target both in vitro and in vivo. Exosomes from DDP-resistant lung cancer cells A549 can alter other lung cancer cells' sensitivity to DDP in exosomal miR-100-5p-dependent manner. Our study provides new insights into the molecular mechanism of DDP resistance in lung cancer.

  7. Identification of archaeal proteins that affect the exosome function in vitro

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    Palhano Fernando L

    2010-05-01

    Full Text Available Abstract Background The archaeal exosome is formed by a hexameric RNase PH ring and three RNA binding subunits and has been shown to bind and degrade RNA in vitro. Despite extensive studies on the eukaryotic exosome and on the proteins interacting with this complex, little information is yet available on the identification and function of archaeal exosome regulatory factors. Results Here, we show that the proteins PaSBDS and PaNip7, which bind preferentially to poly-A and AU-rich RNAs, respectively, affect the Pyrococcus abyssi exosome activity in vitro. PaSBDS inhibits slightly degradation of a poly-rA substrate, while PaNip7 strongly inhibits the degradation of poly-A and poly-AU by the exosome. The exosome inhibition by PaNip7 appears to depend at least partially on its interaction with RNA, since mutants of PaNip7 that no longer bind RNA, inhibit the exosome less strongly. We also show that FITC-labeled PaNip7 associates with the exosome in the absence of substrate RNA. Conclusions Given the high structural homology between the archaeal and eukaryotic proteins, the effect of archaeal Nip7 and SBDS on the exosome provides a model for an evolutionarily conserved exosome control mechanism.

  8. Saliva exosomes from pancreatic tumor-bearing mice modulate NK cell phenotype and antitumor cytotoxicity.

    Science.gov (United States)

    Katsiougiannis, Stergios; Chia, David; Kim, Yong; Singh, Ram P; Wong, David T W

    2017-03-01

    Tumor exosomes are emerging as antitumor immunity regulators; however, their effects on secondary exosome secretion by distal organs have not been explored. We have previously demonstrated that suppression of exosomes at the distal tumor site of pancreatic ductal adenocarcinoma (PDAC) ablated the development of salivary biomarker profile. Here, we explore the function of salivary exosomes from tumor-bearing mice in immune surveillance. We provide evidence that salivary exosomes from mice with PDAC exhibit a suppressive effect that results in reduced tumor-killing capacity by NK cells. Salivary exosomes from mice with PDAC where pancreatic tumors were engineered to suppress exosome biogenesis failed to suppress NK cell cytotoxic potential against tumor cells, as opposed to salivary exosomes from mice with PDAC with normal tumor exosome biogenesis. These results reveal an important and previously unknown mechanism of antitumor immune regulation and provide new insights into our understanding of the alterations of this biofluid during tumor development.-Katsiougiannis, S., Chia, D., Kim, Y., Singh, R. P., Wong, D. T. W. Saliva exosomes from pancreatic tumor-bearing mice modulate NK cell phenotype and antitumor cytotoxicity. © FASEB.

  9. Neutrophil-Derived Exosomes: A New Mechanism Contributing to Airway Smooth Muscle Remodeling.

    Science.gov (United States)

    Vargas, Amandine; Roux-Dalvai, Florence; Droit, Arnaud; Lavoie, Jean-Pierre

    2016-09-01

    Neutrophils infiltrate the airways of patients with asthma of all severities, yet their role in the pathogenesis of asthma and their contribution to airway remodeling is largely unknown. We hypothesized that neutrophils modulate airway smooth muscle (ASM) proliferation in asthma by releasing bioactive exosomes. These newly discovered nano-sized vesicles have the capacity to modulate immune responses, cell migration, cell differentiation, and other aspects of cell-to-cell communication. The aim of the study is to determine whether bioactive exosomes are released by neutrophils, and, if so, characterize their proteomic profile and evaluate their capacity to modulate ASM cell proliferation. Exosomes were isolated from equine neutrophil supernatants by differential centrifugation and filtration methods, followed by size-exclusion chromatography. Nanovesicles were characterized using electron microscopy, particle size determination, and proteomic analyses. Exosomes were cocultured with ASM cells and analyzed for exosome internalization by confocal microscopy. ASM proliferation was measured using an impedance-based system. Neutrophils release exosomes that have characteristic size, morphology, and exosomal markers. We identified 271 proteins in exosomes from both LPS and unstimulated neutrophils, and 16 proteins that were differentially expressed, which carried proteins associated with immune response and positive regulation of cell communication. Furthermore, neutrophil-derived exosomes were rapidly internalized by ASM cells and altered their proliferative properties. Upon stimulation of LPS, neutrophil-derived exosomes can enhance the proliferation of ASM cells and could therefore play an important role in the progression of asthma and promoting airway remodeling in severe and corticosteroid-insensitive patients with asthma.

  10. Exosomes Derived from Human Umbilical Cord Mesenchymal Stem Cells Relieve Acute Myocardial Ischemic Injury

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    Yuanyuan Zhao

    2015-01-01

    Full Text Available This study is aimed at investigating whether human umbilical cord mesenchymal stem cell- (hucMSC- derived exosomes (hucMSC-exosomes have a protective effect on acute myocardial infarction (AMI. Exosomes were characterized under transmission electron microscopy and the particles of exosomes were further examined through nanoparticle tracking analysis. Exosomes (400 μg protein were intravenously administrated immediately following ligation of the left anterior descending (LAD coronary artery in rats. Cardiac function was evaluated by echocardiography and apoptotic cells were counted using TUNEL staining. The cardiac fibrosis was assessed using Masson’s trichrome staining. The Ki67 positive cells in ischemic myocardium were determined using immunohistochemistry. The effect of hucMSC-exosomes on blood vessel formation was evaluated through tube formation and migration of human umbilical vein endothelial cells (EA.hy926 cells. The results indicated that ligation of the LAD coronary artery reduced cardiac function and induced cardiomyocyte apoptosis. Administration of hucMSC-exosomes significantly improved cardiac systolic function and reduced cardiac fibrosis. Moreover, hucMSC-exosomes protected myocardial cells from apoptosis and promoted the tube formation and migration of EA.hy926 cells. It is concluded that hucMSC-exosomes improved cardiac systolic function by protecting myocardial cells from apoptosis and promoting angiogenesis. These effects of hucMSC-exosomes might be associated with regulating the expression of Bcl-2 family.

  11. Exosomes from Osteosarcoma and normal osteoblast differ in proteomic cargo and immunomodulatory effects on T cells.

    Science.gov (United States)

    Troyer, Ryan M; Ruby, Carl E; Goodall, Cheri P; Yang, Liping; Maier, Claudia S; Albarqi, Hassan A; Brady, Jacqueline V; Bathke, Kallan; Taratula, Oleh; Mourich, Dan; Bracha, Shay

    2017-09-15

    Canine osteosarcoma (OSA) is the most common cancer of the appendicular skeleton and is associated with high metastatic rate to the lungs and poor prognosis. Recent studies have shown the impact of malignant-derived exosomes on immune cells and the facilitation of immune evasion. In the current study, we have characterized the proteomic profile of exosomes derived from healthy osteoblasts and osteosarcoma cell lines. We investigated the direct impact of these exosomes on healthy T cells. Proteomic cargo of the malignant exosomes was markedly different from osteoblastic exosomes and contained immunosuppressive proteins including TGF-β, α fetoprotein and heat shock proteins. OSA exosomes directly attenuated the rate of T cell proliferation, increased a regulatory (FoxP3+) CD4+ phenotype and diminished the expression of the activation marker CD25+ on CD8+ cells. Exosomes of osteoblasts also demonstrated a direct impact on T cells, but to a lesser degree. Osteosarcoma-derived exosomes compared to normal osteoblasts contain an immunomodulatory cargo, which reduced the rate of T cell proliferation and promoted T regulatory phenotype. Osteoblast-derived exosomes can also reduce T cell activity, but to lesser degree compared to OSA exosomes and without promoting a T regulatory phenotype. Copyright © 2017 Elsevier Inc. All rights reserved.

  12. Exosome production and its regulation of EGFR during wound healing in renal tubular cells.

    Science.gov (United States)

    Zhou, Xiangjun; Zhang, Wei; Yao, Qisheng; Zhang, Hao; Dong, Guie; Zhang, Ming; Liu, Yutao; Chen, Jian-Kang; Dong, Zheng

    2017-06-01

    Kidney repair following injury involves the reconstitution of a structurally and functionally intact tubular epithelium. Growth factors and their receptors, such as EGFR, are important in the repair of renal tubules. Exosomes are cell-produced small (~100 nm in diameter) vesicles that contain and transfer proteins, lipids, RNAs, and DNAs between cells. In this study, we examined the relationship between exosome production and EGFR activation and the potential role of exosome in wound healing. EGFR activation occurred shortly after scratch wounding in renal tubular cells. Wound repair after scratching was significantly promoted by EGF and suppressed by EGFR inhibitor gefitinib. Interestingly, scratch wounding induced a significant increase of exosome production. The exosome production was decreased by EGF and increased by gefitinib, suggesting a suppressive role of EGFR signaling in exosome production. Conversely, inhibition of exosome release by GW4869 and manumycin A markedly increased EGFR activation and promoted wound healing. Moreover, exosomes derived from scratch-wounding cells could inhibit wound healing. Collectively, the results indicate that wound healing in renal tubular cells is associated with EGFR activation and exosome production. Although EGFR activation promotes wound healing, released exosomes may antagonize EGFR activation and wound healing. Copyright © 2017 the American Physiological Society.

  13. Isolation of exosomes from whole blood by integrating acoustics and microfluidics.

    Science.gov (United States)

    Wu, Mengxi; Ouyang, Yingshi; Wang, Zeyu; Zhang, Rui; Huang, Po-Hsun; Chen, Chuyi; Li, Hui; Li, Peng; Quinn, David; Dao, Ming; Suresh, Subra; Sadovsky, Yoel; Huang, Tony Jun

    2017-10-03

    Exosomes are nanoscale extracellular vesicles that play an important role in many biological processes, including intercellular communications, antigen presentation, and the transport of proteins, RNA, and other molecules. Recently there has been significant interest in exosome-related fundamental research, seeking new exosome-based biomarkers for health monitoring and disease diagnoses. Here, we report a separation method based on acoustofluidics (i.e., the integration of acoustics and microfluidics) to isolate exosomes directly from whole blood in a label-free and contact-free manner. This acoustofluidic platform consists of two modules: a microscale cell-removal module that first removes larger blood components, followed by extracellular vesicle subgroup separation in the exosome-isolation module. In the cell-removal module, we demonstrate the isolation of 110-nm particles from a mixture of micro- and nanosized particles with a yield greater than 99%. In the exosome-isolation module, we isolate exosomes from an extracellular vesicle mixture with a purity of 98.4%. Integrating the two acoustofluidic modules onto a single chip, we isolated exosomes from whole blood with a blood cell removal rate of over 99.999%. With its ability to perform rapid, biocompatible, label-free, contact-free, and continuous-flow exosome isolation, the integrated acoustofluidic device offers a unique approach to investigate the role of exosomes in the onset and progression of human diseases with potential applications in health monitoring, medical diagnosis, targeted drug delivery, and personalized medicine.

  14. A gestational profile of placental exosomes in maternal plasma and their effects on endothelial cell migration.

    Directory of Open Access Journals (Sweden)

    Carlos Salomon

    Full Text Available Studies completed to date provide persuasive evidence that placental cell-derived exosomes play a significant role in intercellular communication pathways that potentially contribute to placentation and development of materno-fetal vascular circulation. The aim of this study was to establish the gestational-age release profile and bioactivity of placental cell-derived exosome in maternal plasma. Plasma samples (n = 20 per pregnant group were obtained from non-pregnant and pregnant women in the first (FT, 6-12 weeks, second (ST, 22-24 weeks and third (TT, 32-38 weeks trimester. The number of exosomes and placental exosome contribution were determined by quantifying immunoreactive exosomal CD63 and placenta-specific marker (PLAP, respectively. The effect of exosomes isolated from FT, ST and TT on endothelial cell migration were established using a real-time, live-cell imaging system (Incucyte. Exosome plasma concentration was more than 50-fold greater in pregnant women than in non-pregnant women (p<0.001. During normal healthy pregnancy, the number of exosomes present in maternal plasma increased significantly with gestational age by more that two-fold (p<0.001. Exosomes isolated from FT, ST and TT increased endothelial cell migration by 1.9±0.1, 1.6±0.2 and 1.3±0.1-fold, respectively compared to the control. Pregnancy is associated with a dramatic increase in the number of exosomes present in plasma and maternal plasma exosomes are bioactive. While the role of placental cell-derived exosome in regulating maternal and/or fetal vascular responses remains to be elucidated, changes in exosome profile may be of clinical utility in the diagnosis of placental dysfunction.

  15. Urinary Exosomes: The Potential for Biomarker Utility, Intercellular Signaling and Therapeutics in Urological Malignancy.

    Science.gov (United States)

    Franzen, Carrie A; Blackwell, Robert H; Foreman, Kimberly E; Kuo, Paul C; Flanigan, Robert C; Gupta, Gopal N

    2016-05-01

    Exosomes are small secreted vesicles that contain proteins, mRNA and miRNA with the potential to alter signaling pathways in recipient cells. While exosome research has flourished, few publications have specifically considered the role of genitourinary cancer shed exosomes in urine, their implication in disease progression and their usefulness as noninvasive biomarkers. In this review we examined the current literature on the role of exosomes in intercellular communication and as biomarkers, and their potential as delivery vehicles for therapeutic applications in bladder, prostate and renal cancer. We searched PubMed® and Google® with the key words prostate cancer, bladder cancer, kidney cancer, exosomes, microvesicles and urine. Relevant articles, including original research studies and reviews, were selected based on contents. A review of this literature was generated. Cancer exosomes can be isolated from urine using various techniques. Cancer cells have been found to secrete more exosomes than normal cells. These exosomes have a role in cellular communication by interacting with and depositing their cargo in target cells. Bladder, prostate and renal cancer exosomes have been shown to enhance migration, invasion and angiogenesis. These exosomes have also been shown to increase proliferation, confer drug resistance and promote immune evasion. Urinary exosomes can be isolated from bladder, kidney and prostate cancer. They serve as a potential reservoir for biomarker identification. Exosomes also have potential for therapeutics as siRNA or pharmacological agents can be loaded into exosomes. Copyright © 2016 American Urological Association Education and Research, Inc. Published by Elsevier Inc. All rights reserved.

  16. Tumor-Derived Exosomes and Their Role in Tumor-Induced Immune Suppression

    Directory of Open Access Journals (Sweden)

    Theresa L. Whiteside

    2016-10-01

    Full Text Available Tumor-derived exosomes (TEX are emerging as critical components of an intercellular information network between the tumor and the host. The tumor escapes from the host immune system by using a variety of mechanisms designed to impair or eliminate anti-tumor immunity. TEX carrying a cargo of immunoinhibitory molecules and factors represent one such mechanism. TEX, which are present in all body fluids of cancer patients, deliver negative molecular or genetic signals to immune cells re-programming their functions. Although TEX can also stimulate immune activity, in the microenvironments dominated by the tumor, TEX tend to mediate immune suppression thus promoting tumor progression. The TEX content, in part resembling that of the parent cell, may serve as a source of cancer biomarkers. TEX also interfere with immune therapies. A better understanding of TEX and their contribution to cancer progression and cancer patients’ response to immune therapies represents a challenging new field of investigation.

  17. Exosomes and their Application in Biomedical Field: Difficulties and Advantages.

    Science.gov (United States)

    Rezaie, Jafar; Ajezi, Saeed; Avci, Çığır Biray; Karimipour, Mohammad; Geranmayeh, Mohammad Hossein; Nourazarian, Alireza; Sokullu, Emel; Rezabakhsh, Aysa; Rahbarghazi, Reza

    2017-05-11

    The science of gene therapy has experienced a controversial history. At first, the initial concept that various disorders become curable by gene transferring was very exciting and challengeable. However, the problems and difficulties related to emerging techniques and unwanted side effects seen in some patients who have undergone gene therapy make some questions against the safety of novel molecular medicine approach. In line with this statement, discovery and developing a good bio-vector possessing low toxicity and high efficiency rate are the most important issues in gene therapy field. Introducing exosomes as vectors for gene delivery gives us a new opportunity in gene-based therapy. Exosomes, ranging from 30 to 120 nm in diameter, have unique lipid and protein composition. These nanostructures participate in cell-to-cell cross-talk, regulation of immune system, and the transport of genetic material. Besides the inherent potency of exosomes in gene therapy, a better understanding of their biology, characteristics, production, targeting, and cargo loading still need to be elucidated. In the current review, we exclusively focused on the various facets of exosomes and their importance as a bio-shuttle in gene therapy.

  18. Exosomic microRNAs in the tumor microenvironment

    Directory of Open Access Journals (Sweden)

    Paolo eNeviani

    2015-07-01

    Full Text Available Dissecting the cross talk between tumor cells and tumor microenvironment is quickly becoming the new frontier in cancer research. It is now widely accepted that cancer cells can exert a profound influence over their surroundings, by changing the microenvironment from a normal to a tumor-supportive state that allows for sustained tumor-growth, invasion and drug-resistance. Extracellular vesicles, especially exosomes, are recognized as a new category of intercellular communicator and they are emerging as of primary importance in controlling the interplay between the tumor and its environment. Exosomes derived from cancer cells or from cells of the tumor microenvironment allow for the horizontal transfer of information by virtue of their cargo, made of functional proteins and nucleic acids that are specifically sorted and loaded in exosomes during their biogenesis. In this review we will discuss the current knowledge regarding the role invested by microRNAs, a family of short non-coding RNAs frequently deregulated in malignancies and present in exosomes, in shaping the microenvironment in a cancer-dependent manner.

  19. Identification of a Nuclear Exosome Decay Pathway for Processed Transcripts

    DEFF Research Database (Denmark)

    Meola, Nicola; Domanski, Michal; Karadoulama, Evdoxia

    2016-01-01

    , the Zn-finger protein ZCCHC8, and the RNA-binding factor RBM7. NEXT primarily targets early and unprocessed transcripts, which demands a rationale for how the nuclear exosome recognizes processed RNAs. Here, we describe the poly(A) tail exosome targeting (PAXT) connection, which comprises the ZFC3H1 Zn......The RNA exosome is fundamental for the degradation of RNA in eukaryotic nuclei. Substrate targeting is facilitated by its co-factor Mtr4p/hMTR4, which links to RNA-binding protein adaptors. One example is the trimeric human nuclear exosome targeting (NEXT) complex, which is composed of hMTR4......-knuckle protein as a central link between hMTR4 and the nuclear poly(A)-binding protein PABPN1. Individual depletion of ZFC3H1 and PABPN1 results in the accumulation of common transcripts that are generally both longer and more extensively polyadenylated than NEXT substrates. Importantly, ZFC3H1/PABPN1 and ZCCHC8...

  20. Exosomes as potent cell-free peptide-based vaccine. II. Exosomes in CpG adjuvants efficiently prime naive Tc1 lymphocytes leading to tumor rejection.

    NARCIS (Netherlands)

    Chaput, N.; Schartz, N.E.; Andre, F.; Taieb, J.; Novault, S.; Bonnaventure, P.; Aubert, N.; Bernard, J.; Lemonnier, F.; Merad, M.; Adema, G.J.; Adams, M.; Ferrantini, M.; Carpentier, A.F.; Escudier, B.; Tursz, T.; Angevin, E.; Zitvogel, L.

    2004-01-01

    Ideal vaccines should be stable, safe, molecularly defined, and out-of-shelf reagents efficient at triggering effector and memory Ag-specific T cell-based immune responses. Dendritic cell-derived exosomes could be considered as novel peptide-based vaccines because exosomes harbor a discrete set of

  1. Clinical implications of carcinoembryonic antigen distribution in serum exosomal fraction-Measurement by ELISA.

    Directory of Open Access Journals (Sweden)

    Shozo Yokoyama

    Full Text Available Serum exosomal proteins have great potential as indicators of disease status in cancer, inflammatory or metabolic diseases. The association of a fraction of various serum proteins such as carcinoembryonic antigen (CEA with circulating exosomes has been debated. The establishment of a method to measure the exosomal fraction of such proteins might help resolve this controversy. The use of enzyme-linked immunosorbent assays (ELISAs to measure serum exosomal molecules, for example CEA, is rare in research laboratories and totally absent in clinical biology. In this study, we optimized a method for assessment of serum exosomal molecules combining a treatment by volume-excluding polymers to isolate the exosomes, their subsequent solubilization in an assay buffer and ELISA.One hundred sixteen consecutive patients with colorectal cancer were enrolled for this study between June 2015 and June 2016 at Wakayama Medical University Hospital (WMUH. Whole blood samples were collected from patients during surgery. Exosomes were isolated using the ExoQuick reagent, solubilized in an assay buffer and subjected to CEA detection by ELISA. The procedure of serum exosome isolation and the formulation of the assay buffer used for the ELISA were optimized in order to improve the sensitivity and specificity of the assay.A five-fold increase in the concentration of the exosomes in the assay buffer (using initial serum volume as a reference and the addition of bovine serum albumin (BSA resulted in more accurate measurements of the serum exosomal CEA. The thawing temperature of frozen serum samples before exosome extraction was also optimized. A validation study that included one hundred sixteen patients with colorectal cancer demonstrated that serum exosomal CEA from samples thawed at 25°C exhibited a better AUC value, sensitivity, and specificity as well as a more correct classification than serum CEA.We optimized an easy and rapid detection method for assessment of

  2. Breast cancer exosome-like microvesicles and salivary gland cells interplay alters salivary gland cell-derived exosome-like microvesicles in vitro.

    Directory of Open Access Journals (Sweden)

    Chang S Lau

    Full Text Available Saliva is a useful biofluid for the early detection of disease, but how distal tumors communicate with the oral cavity and create disease-specific salivary biomarkers remains unclear. Using an in vitro breast cancer model, we demonstrated that breast cancer-derived exosome-like microvesicles are capable of interacting with salivary gland cells, altering the composition of their secreted exosome-like microvesicles. We found that the salivary gland cells secreted exosome-like microvesicles encapsulating both protein and mRNA. We also showed that the interaction with breast cancer-derived exosome-like microvesicles communicated and activated the transcriptional machinery of the salivary gland cells. Thus, the interaction altered the composition of the salivary gland cell-derived exosome-like microvesicles on both the transcriptomically and proteomically.

  3. The characterization of exosomes from biological fluids of patients with different types of cancer

    Science.gov (United States)

    Yunusova, N. V.; Tamkovich, S. N.; Stakheeva, M. N.; Grigor'eva, A. A.; Somov, A. K.; Tugutova, E. A.; Kolomiets, L. A.; Molchanov, S. V.; Afanas'ev, S. G.; Kakurina, G. V.; Choinzonov, E. L.; Kondakova, I. V.

    2017-09-01

    Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The purpose of the study was to isolate, identify and analyze the total content of exosomes in biological fluids. The exosomes from the plasma and ascites samples of the patients with ovarian cancer, from the blood plasma of the patients with colorectal and head and neck squamous cell cancer as well as from the blood plasma of healthy donors were characterized using transmission electron microscopy and flow cytometry. The subpopulations of the exosomes in the biological fluids of the patients with different types of cancer were similar, but the protein concentrations of exosomes were different. In this paper we present the methodological approaches allowing us to obtain high quality exosome preparations from biological fluids.

  4. The biology, function and clinical implications of exosomes in lung cancer.

    Science.gov (United States)

    Zhou, Li; Lv, Tangfeng; Zhang, Qun; Zhu, Qingqing; Zhan, Ping; Zhu, Suhua; Zhang, Jianya; Song, Yong

    2017-10-28

    Exosomes are 30-100 nm small membrane vesicles of endocytic origin that are secreted by all types of cells, and can also be found in various body fluids. Increasing evidence implicates that exosomes confer stability and can deliver their cargos such as proteins and nucleic acids to specific cell types, which subsequently serve as important messengers and carriers in lung carcinogenesis. Here, we describe the biogenesis and components of exosomes mainly in lung cancer, we summarize their function in lung carcinogenesis (epithelial mesenchymal transition, oncogenic cell transformation, angiogenesis, metastasis and immune response in tumor microenvironment), and importantly we focus on the clinical potential of exosomes as biomarkers and therapeutics in lung cancer. In addition, we also discuss current challenges that might impede the clinical use of exosomes. Further studies on the functional roles of exosomes in lung cancer requires thorough research. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. [Advances in the research of effects of exosomes derived from stem cells on wound repair].

    Science.gov (United States)

    Li, M Y; Liu, D W; Mao, Y G

    2017-03-20

    Exosomes are nano-vesicles released by many kinds of cells. Exosomes play a significant role in cell-to-cell communication and substance transportation through direct effect of signaling molecules on the cell membrane surface, intracellular regulation of cellular content during membrane fusion, or regulation of release of various bioactive molecules. Several studies have reported that culture supernatant of stem cells has some related exosomes to take part in wound repair. The secretion of exosomes is depended on the source and the physiological and pathological condition of deriving cells. How to stimulate the stem cells to produce exosomes maximally and their clinical application are worthy to explore. In this review, we summarize the biological function and application of exosomes derived from stem cells in wound repair.

  6. Extracellular Vesicles as Biomarkers and Therapeutics in Dermatology: A Focus on Exosomes.

    Science.gov (United States)

    McBride, Jeffrey D; Rodriguez-Menocal, Luis; Badiavas, Evangelos V

    2017-08-01

    Extracellular vesicles (exosomes, microvesicles, and apoptotic bodies) are ubiquitous in human tissues, circulation, and body fluids. Of these vesicles, exosomes are of growing interest among investigators across multiple fields, including dermatology. The characteristics of exosomes, their associated cargo (nucleic acids, proteins, and lipids), and downstream functions are vastly different, depending on the cell origin. Here, we review concepts in extracellular vesicle biology, with a focus on exosomes, highlighting recent studies in the field of dermatology. Furthermore, we highlight emerging technical issues associated with isolating and measuring exosomes. Extracellular vesicles, including exosomes, have immediate potential for serving as biomarkers and therapeutics in dermatology over the next decade. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  7. New evidence that a large proportion of human blood plasma cell-free DNA is localized in exosomes.

    Science.gov (United States)

    Fernando, M Rohan; Jiang, Chao; Krzyzanowski, Gary D; Ryan, Wayne L

    2017-01-01

    Cell-free DNA (cfDNA) in blood is used as a source of genetic material for noninvasive prenatal and cancer diagnostic assays in clinical practice. Recently we have started a project for new biomarker discovery with a view to developing new noninvasive diagnostic assays. While reviewing literature, it was found that exosomes may be a rich source of biomarkers, because exosomes play an important role in human health and disease. While characterizing exosomes found in human blood plasma, we observed the presence of cfDNA in plasma exosomes. Plasma was obtained from blood drawn into K3EDTA tubes. Exosomes were isolated from cell-free plasma using a commercially available kit. Sizing and enumeration of exosomes were done using electron microscopy and NanoSight particle counter. NanoSight and confocal microscopy was used to demonstrate the association between dsDNA and exosomes. DNA extracted from plasma and exosomes was measured by a fluorometric method and a droplet digital PCR (ddPCR) method. Size of extracellular vesicles isolated from plasma was heterogeneous and showed a mean value of 92.6 nm and a mode 39.7 nm. A large proportion of extracellular vesicles isolated from plasma were identified as exosomes using a fluorescence probe specific for exosomes and three protein markers, Hsp70, CD9 and CD63, that are commonly used to identify exosome fraction. Fluorescence dye that stain dsDNA showed the association between exosomes and dsDNA. Plasma cfDNA concentration analysis showed more than 93% of amplifiable cfDNA in plasma is located in plasma exosomes. Storage of a blood sample showed significant increases in exosome count and exosome DNA concentration. This study provide evidence that a large proportion of plasma cfDNA is localized in exosomes. Exosome release from cells is a metabolic energy dependent process, thus suggesting active release of cfDNA from cells as a source of cfDNA in plasma.

  8. A hyphenated microLC-Q-TOF-MS platform for exosomal lipidomics investigations: application to RCC urinary exosomes.

    Science.gov (United States)

    Del Boccio, Piero; Raimondo, Francesca; Pieragostino, Damiana; Morosi, Lavinia; Cozzi, Gabriele; Sacchetta, Paolo; Magni, Fulvio; Pitto, Marina; Urbani, Andrea

    2012-02-01

    Urinary exosomes are released from every renal epithelial cell type facing the urinary space and therefore, they may carry molecular markers of renal dysfunction and structural injury. Here, we present a hyphenated microLC-Q-TOF-MS platform for lipidomics studies applied to investigate the urinary exosome lipid repertoire. Lipids were separated by reversed-phase chromatography using a linear gradient of formic acid 0.2% and tetrahydrofuran, in 40 min of analysis. Features (m/z with associated own retention time) were extracted by MarkerLynx(TM) (Waters) and processed, demonstrating good analytical performance in terms of repeatability and mass accuracy of the microLC Q-TOF MS platform. In particular, a stable retention time (RSD less than 4%) and relative intensity (RSD from 2.9% to 11%) were observed. Moreover, the method takes advantages by the use of a lock spray interface (Waters) that allows readjusting the m/z data after acquisition, obtaining inaccuracy below 6 ppm in measuring the m/z value of the reference compound during chromatographic run. The method was employed in a preliminary application to perform comparative analysis from healthy control subjects and renal cell carcinoma (RCC) patients, in order to possibly highlight differences in lipid composition to be exploited as potential tumor biomarker. Differential lipid composition in RCC urinary exosomes was achieved and tentatively identified by accurate mass, providing a preliminary indication of a relationship between lipid composition of urinary exosomes and RCC disease. Among the total features significantly different in RCC exosomes, the ion at m/z 502.3 was taken as an example for molecular confirmation by MS/MS fragmentation analysis. Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Exosomes Derived From Natural Killer Cells Exert Therapeutic Effect in Melanoma

    OpenAIRE

    Zhu, Liya; Kalimuthu, Senthilkumar; Gangadaran, Prakash; Oh, Ji Min; Lee, Ho Won; Baek, Se hwan; Jeong, Shin Young; Lee, Sang-Woo; Lee, Jaetae; Ahn, Byeong-Cheol

    2017-01-01

    Objective: Exosomes are nanovesicles that are released from normal and tumor cells and are detectable in cell culture supernatant and human biological fluids. Although previous studies have explored exosomes released from cancer cells, little is understood regarding the functions of exosomes released by normal cells. Natural killer (NK) cells display rapid immunity to metastatic or hematological malignancies, and efforts have been undertaken to clinically exploit the antitumor properties of N...

  10. Exosomes as Novel microRNA-Delivery Vehicles to Modulate Prostate Cancer Progression

    Science.gov (United States)

    2015-10-01

    AWARD NUMBER: W81XWH-14-1-0548 TITLE: Exosomes as Novel microRNA-Delivery Vehicles to Modulate Prostate Cancer Progression PRINCIPAL...Sep 2015 4. TITLE AND SUBTITLE Exosomes as Novel microRNA-Delivery Vehicles to Modulate Prostate Cancer Progression 5a. CONTRACT NUMBER 5b. GRANT...they are produced, but can also signal intercellularly to other cells and tissues at distant sites via exosomal transport. We hypothesize that miRNAs

  11. Exosomes Derived from HIV-1-infected Cells Contain Trans-activation Response Element RNA*

    Science.gov (United States)

    Narayanan, Aarthi; Iordanskiy, Sergey; Das, Ravi; Van Duyne, Rachel; Santos, Steven; Jaworski, Elizabeth; Guendel, Irene; Sampey, Gavin; Dalby, Elizabeth; Iglesias-Ussel, Maria; Popratiloff, Anastas; Hakami, Ramin; Kehn-Hall, Kylene; Young, Mary; Subra, Caroline; Gilbert, Caroline; Bailey, Charles; Romerio, Fabio; Kashanchi, Fatah

    2013-01-01

    Exosomes are nano-sized vesicles produced by healthy and virus-infected cells. Exosomes derived from infected cells have been shown to contain viral microRNAs (miRNAs). HIV-1 encodes its own miRNAs that regulate viral and host gene expression. The most abundant HIV-1-derived miRNA, first reported by us and later by others using deep sequencing, is the trans-activation response element (TAR) miRNA. In this study, we demonstrate the presence of TAR RNA in exosomes from cell culture supernatants of HIV-1-infected cells and patient sera. TAR miRNA was not in Ago2 complexes outside the exosomes but enclosed within the exosomes. We detected the host miRNA machinery proteins Dicer and Drosha in exosomes from infected cells. We report that transport of TAR RNA from the nucleus into exosomes is a CRM1 (chromosome region maintenance 1)-dependent active process. Prior exposure of naive cells to exosomes from infected cells increased susceptibility of the recipient cells to HIV-1 infection. Exosomal TAR RNA down-regulated apoptosis by lowering Bim and Cdk9 proteins in recipient cells. We found 104–106 copies/ml TAR RNA in exosomes derived from infected culture supernatants and 103 copies/ml TAR RNA in the serum exosomes of highly active antiretroviral therapy-treated patients or long term nonprogressors. Taken together, our experiments demonstrated that HIV-1-infected cells produced exosomes that are uniquely characterized by their proteomic and RNA profiles that may contribute to disease pathology in AIDS. PMID:23661700

  12. CD109 is a component of exosome secreted from cultured cells

    Energy Technology Data Exchange (ETDEWEB)

    Sakakura, Hiroki [Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya (Japan); Mii, Shinji [Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Hagiwara, Sumitaka [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya (Japan); Department of Head and Neck Surgery, Aichi Cancer Center Hospital, Nagoya (Japan); Kato, Takuya [Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Yamamoto, Noriyuki; Hibi, Hideharu [Department of Oral and Maxillofacial Surgery, Nagoya University Graduate School of Medicine, Nagoya (Japan); Takahashi, Masahide, E-mail: mtakaha@med.nagoya-u.ac.jp [Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Murakumo, Yoshiki, E-mail: murakumo@med.kitasato-u.ac.jp [Department of Pathology, Nagoya University Graduate School of Medicine, Nagoya (Japan); Department of Pathology, Kitasato University School of Medicine, Sagamihara, Kanagawa (Japan)

    2016-01-22

    Exosomes are 50–100-nm-diameter membrane vesicles released from various types of cells. Exosomes retain proteins, mRNAs and miRNAs, which can be transported to surrounding cells. CD109 is a glycosylphosphatidylinositol-anchored glycoprotein, and is released from the cell surface to the culture medium in vitro. Recently, it was reported that secreted CD109 from the cell surface downregulates transforming growth factor-β signaling in human keratinocytes. In this study, we revealed that CD109 is a component of the exosome in conditioned medium. FLAG-tagged human CD109 (FLAG-CD109) in conditioned medium secreted from HEK293 cells expressing FLAG-CD109 (293/FLAG-CD109) was immunoprecipitated with anti-FLAG affinity gel, and the co-precipitated proteins were analyzed by mass spectrometry and western blotting. Exosomal proteins were associated with CD109. We revealed the presence of CD109 in exosome fractions from conditioned medium of 293/FLAG-CD109. Moreover, the localization of CD109 in the exosome was demonstrated using immuno-electron microscopy. When we used HEK293 cells expressing FLAG-tagged truncated CD109, which does not contain the C-terminal region, the association of truncated CD109 with exosomes was not detected in conditioned medium. These findings indicate that CD109 is an exosomal protein and that the C-terminal region of CD109 is required for its presence in the exosome. - Highlights: • CD109 is an exosomal protein. • The C-terminal region of CD109 is required for its presence in the exosome. • Part of the secreted CD109 is present in the exosome-free fraction in the conditioned medium.

  13. Plasma-derived exosomal survivin, a plausible biomarker for early detection of prostate cancer.

    Directory of Open Access Journals (Sweden)

    Salma Khan

    Full Text Available Survivin is expressed in prostate cancer (PCa, and its downregulation sensitizes PCa cells to chemotherapeutic agents in vitro and in vivo. Small membrane-bound vesicles called exosomes, secreted from the endosomal membrane compartment, contain RNA and protein that they readily transport via exosome internalization into recipient cells. Recent progress has shown that tumor-derived exosomes play multiple roles in tumor growth and metastasis and may produce these functions via immune escape, tumor invasion and angiogenesis. Furthermore, exosome analysis may provide novel biomarkers to diagnose or monitor PCa treatment.Exosomes were purified from the plasma and serum from 39 PCa patients, 20 BPH patients, 8 prostate cancer recurrent and 16 healthy controls using ultracentrifugation and their quantities and qualities were quantified and visualized from both the plasma and the purified exosomes using ELISA and Western blotting, respectively.Survivin was significantly increased in the tumor-derived samples, compared to those from BPH and controls with virtually no difference in the quantity of Survivin detected in exosomes collected from newly diagnosed patients exhibiting low (six or high (nine Gleason scores. Exosome Survivin levels were also higher in patients that had relapsed on chemotherapy compared to controls.These studies demonstrate that Survivin exists in plasma exosomes from both normal, BPH and PCa subjects. The relative amounts of exosomal Survivin in PCa plasma was significantly higher than in those with pre-inflammatory BPH and control plasma. This differential expression of exosomal Survivin was seen with both newly diagnosed and advanced PCa subjects with high or low-grade cancers. Analysis of plasma exosomal Survivin levels may offer a convenient tool for diagnosing or monitoring PCa and may, as it is elevated in low as well as high Gleason scored samples, be used for early detection.

  14. Radiolabeled Exosomes for the Early Detection of Metastases and to Predict Breast Cancer Premetastatic Niche

    Science.gov (United States)

    2015-08-31

    breast cancer (BC). This project takes advantage of the breakthrough knowledge in tumor - derived exosome tropism and exploits recent advances in the...exosomes from different cancer models recapitulate the metastatic organotropism of their cell of origin, therefore tumor exosomes could be...radiolabeling protocol in order to achieve a radioactive probe of high specific activity, stability and specificity. Figure 1. Cancer cell-derived

  15. The Complete Exosome Workflow Solution: From Isolation to Characterization of RNA Cargo

    Directory of Open Access Journals (Sweden)

    Jeoffrey Schageman

    2013-01-01

    Full Text Available Exosomes are small (30–150 nm vesicles containing unique RNA and protein cargo, secreted by all cell types in culture. They are also found in abundance in body fluids including blood, saliva, and urine. At the moment, the mechanism of exosome formation, the makeup of the cargo, biological pathways, and resulting functions are incompletely understood. One of their most intriguing roles is intercellular communication—exosomes function as the messengers, delivering various effector or signaling macromolecules between specific cells. There is an exponentially growing need to dissect structure and the function of exosomes and utilize them for development of minimally invasive diagnostics and therapeutics. Critical to further our understanding of exosomes is the development of reagents, tools, and protocols for their isolation, characterization, and analysis of their RNA and protein contents. Here we describe a complete exosome workflow solution, starting from fast and efficient extraction of exosomes from cell culture media and serum to isolation of RNA followed by characterization of exosomal RNA content using qRT-PCR and next-generation sequencing techniques. Effectiveness of this workflow is exemplified by analysis of the RNA content of exosomes derived from HeLa cell culture media and human serum, using Ion Torrent PGM as a sequencing platform.

  16. The characterization of exosome from blood plasma of patients with colorectal cancer

    Science.gov (United States)

    Yunusova, N. V.; Tamkovich, S. N.; Stakheeva, M. N.; Afanas'ev, S. G.; Frolova, A. Y.; Kondakova, I. V.

    2016-08-01

    Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of "International Society for Extracellular Vesicles". The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. The results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.

  17. Involvement of multiple myeloma cell-derived exosomes in osteoclast differentiation

    Science.gov (United States)

    Raimondi, Lavinia; De Luca, Angela; Amodio, Nicola; Manno, Mauro; Raccosta, Samuele; Taverna, Simona; Bellavia, Daniele; Naselli, Flores; Fontana, Simona; Schillaci, Odessa; Giardino, Roberto; Fini, Milena; Tassone, Pierfrancesco; Santoro, Alessandra; De Leo, Giacomo; Giavaresi, Gianluca; Alessandro, Riccardo

    2015-01-01

    Bone disease is the most frequent complication in multiple myeloma (MM) resulting in osteolytic lesions, bone pain, hypercalcemia and renal failure. In MM bone disease the perfect balance between bone-resorbing osteoclasts (OCs) and bone-forming osteoblasts (OBs) activity is lost in favour of OCs, thus resulting in skeletal disorders. Since exosomes have been described for their functional role in cancer progression, we here investigate whether MM cell-derived exosomes may be involved in OCs differentiation. We show that MM cells produce exosomes which are actively internalized by Raw264.7 cell line, a cellular model of osteoclast formation. MM cell-derived exosomes positively modulate pre-osteoclast migration, through the increasing of CXCR4 expression and trigger a survival pathway. MM cell-derived exosomes play a significant pro-differentiative role in murine Raw264.7 cells and human primary osteoclasts, inducing the expression of osteoclast markers such as Cathepsin K (CTSK), Matrix Metalloproteinases 9 (MMP9) and Tartrate-resistant Acid Phosphatase (TRAP). Pre-osteoclast treated with MM cell-derived exosomes differentiate in multinuclear OCs able to excavate authentic resorption lacunae. Similar results were obtained with exosomes derived from MM patient's sera. Our data indicate that MM-exosomes modulate OCs function and differentiation. Further studies are needed to identify the OCs activating factors transported by MM cell-derived exosomes. PMID:25944696

  18. Defining the purity of exosomes required for diagnostic profiling of small RNA suitable for biomarker discovery.

    Science.gov (United States)

    Quek, Camelia; Bellingham, Shayne A; Jung, Chol-Hee; Scicluna, Benjamin J; Shambrook, Mitch C; Sharples, Robyn A; Cheng, Lesley; Hill, Andrew F

    2017-02-01

    Small non-coding RNAs (ncRNA), including microRNAs (miRNA), enclosed in exosomes are being utilised for biomarker discovery in disease. Two common exosome isolation methods involve differential ultracentrifugation or differential ultracentrifugation coupled with Optiprep gradient fractionation. Generally, the incorporation of an Optiprep gradient provides better separation and increased purity of exosomes. The question of whether increased purity of exosomes is required for small ncRNA profiling, particularly in diagnostic and biomarker purposes, has not been addressed and highly debated. Utilizing an established neuronal cell system, we used next-generation sequencing to comprehensively profile ncRNA in cells and exosomes isolated by these 2 isolation methods. By comparing ncRNA content in exosomes from these two methods, we found that exosomes from both isolation methods were enriched with miRNAs and contained a diverse range of rRNA, small nuclear RNA, small nucleolar RNA and piwi-interacting RNA as compared with their cellular counterparts. Additionally, tRNA fragments (30-55 nucleotides in length) were identified in exosomes and may act as potential modulators for repressing protein translation. Overall, the outcome of this study confirms that ultracentrifugation-based method as a feasible approach to identify ncRNA biomarkers in exosomes.

  19. Exosomes in Human Immunodeficiency Virus Type I Pathogenesis: Threat or Opportunity?

    Directory of Open Access Journals (Sweden)

    Sin-Yeang Teow

    2016-01-01

    Full Text Available Nanometre-sized vesicles, also known as exosomes, are derived from endosomes of diverse cell types and present in multiple biological fluids. Depending on their cellular origins, the membrane-bound exosomes packed a variety of functional proteins and RNA species. These microvesicles are secreted into the extracellular space to facilitate intercellular communication. Collective findings demonstrated that exosomes from HIV-infected subjects share many commonalities with Human Immunodeficiency Virus Type I (HIV-1 particles in terms of proteomics and lipid profiles. These observations postulated that HIV-resembled exosomes may contribute to HIV pathogenesis. Interestingly, recent reports illustrated that exosomes from body fluids could inhibit HIV infection, which then bring up a new paradigm for HIV/AIDS therapy. Accumulative findings suggested that the cellular origin of exosomes may define their effects towards HIV-1. This review summarizes the two distinctive roles of exosomes in regulating HIV pathogenesis. We also highlighted several additional factors that govern the exosomal functions. Deeper understanding on how exosomes promote or abate HIV infection can significantly contribute to the development of new and potent antiviral therapeutic strategy and vaccine designs.

  20. Lymphatic transport of exosomes as a rapid route of information dissemination to the lymph node.

    Science.gov (United States)

    Srinivasan, Swetha; Vannberg, Fredrik O; Dixon, J Brandon

    2016-04-18

    It is well documented that cells secrete exosomes, which can transfer biomolecules that impact recipient cells' functionality in a variety of physiologic and disease processes. The role of lymphatic drainage and transport of exosomes is as yet unknown, although the lymphatics play critical roles in immunity and exosomes are in the ideal size-range for lymphatic transport. Through in vivo near-infrared (NIR) imaging we have shown that exosomes are rapidly transported within minutes from the periphery to the lymph node by lymphatics. Using an in vitro model of lymphatic uptake, we have shown that lymphatic endothelial cells actively enhanced lymphatic uptake and transport of exosomes to the luminal side of the vessel. Furthermore, we have demonstrated a differential distribution of exosomes in the draining lymph nodes that is dependent on the lymphatic flow. Lastly, through endpoint analysis of cellular distribution of exosomes in the node, we identified macrophages and B-cells as key players in exosome uptake. Together these results suggest that exosome transfer by lymphatic flow from the periphery to the lymph node could provide a mechanism for rapid exchange of infection-specific information that precedes the arrival of migrating cells, thus priming the node for a more effective immune response.

  1. Characterization of a "TRAMP-like" co-factor of the human RNA exosome

    DEFF Research Database (Denmark)

    Christensen, Marianne Skovgaard; Kristiansen, Maiken Søndergaard; Lubas, Michal Szymon

    exosome, the major 3’-5’ exonuclease complex in human cells. PROMPTs have a lot in common with the yeast Cryptic Unstable Transcripts (CUTs), which are degraded by the concerted effort of the exosome, and its co-factor complex TRAMP (Trf4p/Air1p/Mtr4p). We have identified human proteins with functional...... similarities to components of the yeast TRAMP complex, and show that these are involved in the degradation of PROMPTs. While, these proteins form transient complexes with the exosome, our preliminary results also indicate that complex formation can occur directly with catalytic components of the exosome...

  2. The characterization of exosome from blood plasma of patients with colorectal cancer

    Energy Technology Data Exchange (ETDEWEB)

    Yunusova, N. V., E-mail: Bochkarevanv@oncology.tomsk.ru [Tomsk Cancer Research Institute, Kooperativny Street 5, Tomsk, 634009 (Russian Federation); Siberian State Medical University, Moskovsky Trakt 2, Tomsk, 634050 (Russian Federation); Tamkovich, S. N., E-mail: s.tamk@niboch.nsc.ru [Institute of Chemical Biology and Fundamental Medicine SB RAS, Lavrentiev Avenue 8, Novosibirsk, 630090 (Russian Federation); Novosibirsk State University, Pirogov Street 2, Novosibirsk, 630090 (Russian Federation); Stakheeva, M. N., E-mail: StakheyevaM@oncology.tomsk.ru; Afanas’ev, S. G., E-mail: Afanasievsg@oncology.tomsk.ru; Kondakova, I. V., E-mail: Kondakova@oncology.tomsk.ru [Tomsk Cancer Research Institute, Kooperativny Street 5, Tomsk, 634009 (Russian Federation); Frolova, A. Y., E-mail: Frolovalenya@mail.ru [Siberian State Medical University, Moskovsky Trakt 2, Tomsk, 634050 (Russian Federation)

    2016-08-02

    Exosomes are extracellular membrane structures involved in many physiological and pathological processes including cancerogenesis and metastasis. The clarification of the criteria for exosome isolating and identifying is the purpose of this study. Exosome samples from the plasma of patients with colorectal cancer and healthy donors were examined using transmission electron microscopy and flow cytometry in accordance with the minimum requirements of “International Society for Extracellular Vesicles”. The choice of the method for isolation of exosomes from the blood plasma by ultrafiltration and ultracentrifugation allowed obtaining highly purified samples of exosomes, in which all the structural components were clearly seen. The results obtained with flow cytometry suggest that exosomes of blood plasma from patients with colorectal cancer can be produced by epithelial cells. Moreover, cells produce different types of exosomes, which correspond to different mechanisms in sorting macromolecules in the membrane of multivesicular bodies. Determination of significant differences in the expression of specific exosomal proteins from colorectal cancer patients compared to healthy donors suggests a high diagnostic potential significance of circulating exosomes.

  3. Purification and microRNA profiling of exosomes derived from blood and culture media.

    Science.gov (United States)

    McDonald, Marguerite K; Capasso, Kathryn E; Ajit, Seena K

    2013-06-14

    Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis. Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest. These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

  4. Coronary Artery-Bypass-Graft Surgery Increases the Plasma Concentration of Exosomes Carrying a Cargo of Cardiac MicroRNAs: An Example of Exosome Trafficking Out of the Human Heart with Potential for Cardiac Biomarker Discovery.

    Directory of Open Access Journals (Sweden)

    Costanza Emanueli

    Full Text Available Exosome nanoparticles carry a composite cargo, including microRNAs (miRs. Cultured cardiovascular cells release miR-containing exosomes. The exosomal trafficking of miRNAs from the heart is largely unexplored. Working on clinical samples from coronary-artery by-pass graft (CABG surgery, we investigated if: 1 exosomes containing cardiac miRs and hence putatively released by cardiac cells increase in the circulation after surgery; 2 circulating exosomes and exosomal cardiac miRs correlate with cardiac troponin (cTn, the current "gold standard" surrogate biomarker of myocardial damage.The concentration of exosome-sized nanoparticles was determined in serial plasma samples. Cardiac-expressed (miR-1, miR-24, miR-133a/b, miR-208a/b, miR-210, non-cardiovascular (miR-122 and quality control miRs were measured in whole plasma and in plasma exosomes. Linear regression analyses were employed to establish the extent to which the circulating individual miRs, exosomes and exosomal cardiac miR correlated with cTn-I. Cardiac-expressed miRs and the nanoparticle number increased in the plasma on completion of surgery for up to 48 hours. The exosomal concentration of cardiac miRs also increased after CABG. Cardiac miRs in the whole plasma did not correlate significantly with cTn-I. By contrast cTn-I was positively correlated with the plasma exosome level and the exosomal cardiac miRs.The plasma concentrations of exosomes and their cargo of cardiac miRs increased in patients undergoing CABG and were positively correlated with hs-cTnI. These data provide evidence that CABG induces the trafficking of exosomes from the heart to the peripheral circulation. Future studies are necessary to investigate the potential of circulating exosomes as clinical biomarkers in cardiac patients.

  5. Electrokinetic Evaluation of Individual Exosomes by On-Chip Microcapillary Electrophoresis with Laser Dark-Field Microscopy

    Science.gov (United States)

    Kato, Kei; Kobayashi, Masashi; Hanamura, Nami; Akagi, Takanori; Kosaka, Nobuyoshi; Ochiya, Takahiro; Ichiki, Takanori

    2013-06-01

    Cell-secreted nanovesicles called exosomes are expected as a promising candidate biomarker of various diseases. Toward the future application of exosomes as a disease biomarker for low-invasive diagnostics, challenges remain in the development of sensitive and precise analysis methods for exosomes. In this study, we performed the electrokinetic evaluation of individual exosomes by the combined use of on-chip microcapillary electrophoresis and laser dark-field microscopy. We extracted exosomes from six types of human cell cultured in a serum-free medium by differential ultracentrifugation and their zeta potential (electrophoretic mobility) were evaluated. We demonstrated that the proposed electrophoresis apparatus is particularly suitable for the tracking analysis of the electrophoretic migration of individual exosomes and enables the accurate evaluation of the zeta potential distribution of exosomes, for the first time. From the experimental results, we found that there is a strong correlation between the average zeta potentials of exosomes and their cells of origin.

  6. Soma-to-germline transmission of RNA in mice xenografted with human tumour cells: possible transport by exosomes.

    Directory of Open Access Journals (Sweden)

    Cristina Cossetti

    Full Text Available Mendelian laws provide the universal founding paradigm for the mechanism of genetic inheritance through which characters are segregated and assorted. In recent years, however, parallel with the rapid growth of epigenetic studies, cases of inheritance deviating from Mendelian patterns have emerged. Growing studies underscore phenotypic variations and increased risk of pathologies that are transgenerationally inherited in a non-Mendelian fashion in the absence of any classically identifiable mutation or predisposing genetic lesion in the genome of individuals who develop the disease. Non-Mendelian inheritance is most often transmitted through the germline in consequence of primary events occurring in somatic cells, implying soma-to-germline transmission of information. While studies of sperm cells suggest that epigenetic variations can potentially underlie phenotypic alterations across generations, no instance of transmission of DNA- or RNA-mediated information from somatic to germ cells has been reported as yet. To address these issues, we have now generated a mouse model xenografted with human melanoma cells stably expressing EGFP-encoding plasmid. We find that EGFP RNA is released from the xenografted human cells into the bloodstream and eventually in spermatozoa of the mice. Tumor-released EGFP RNA is associated with an extracellular fraction processed for exosome purification and expressing exosomal markers, in all steps of the process, from the xenografted cancer cells to the spermatozoa of the recipient animals, strongly suggesting that exosomes are the carriers of a flow of information from somatic cells to gametes. Together, these results indicate that somatic RNA is transferred to sperm cells, which can therefore act as the final recipients of somatic cell-derived information.

  7. Leishmania exosomes modulate innate and adaptive immune responses through effects on monocytes and dendritic cells.

    Science.gov (United States)

    Silverman, Judith Maxwell; Clos, Joachim; Horakova, Eva; Wang, Adele Y; Wiesgigl, Martina; Kelly, Isabelle; Lynn, Miriam A; McMaster, W Robert; Foster, Leonard J; Levings, Megan K; Reiner, Neil E

    2010-11-01

    We investigated the properties of leishmania exosomes with respect to influencing innate and adaptive immune responses. Exosomes from Leishmania donovani modulated human monocyte cytokine responses to IFN-γ in a bimodal fashion by promoting IL-10 production and inhibiting that of TNF-α. Moreover, these vesicles were inhibitory with respect to cytokine responses (IL-12p70, TNF-α, and IL-10) by human monocyte-derived dendritic cells. Exosomes from wild-type (WT) L. donovani failed to prime monocyte-derived dendritic cells to drive the differentiation of naive CD4 T cells into IFN-γ-producing Th1 cells. In contrast, vesicles from heat shock protein (HSP)100(-/-) L. donovani showed a gain-of-function and proinflammatory phenotype and promoted the differentiation of naive CD4 lymphocytes into Th1 cells. Proteomic analysis showed that exosomes from WT and HSP100(-/-) leishmania had distinct protein cargo, suggesting that packaging of proteins into exosomes is dependent in part on HSP100. Treatment of C57BL/6 mice with WT L. donovani exosomes prior to challenge with WT organisms exacerbated infection and promoted IL-10 production in the spleen. In contrast, HSP100(-/-) exosomes promoted spleen cell production of IFN-γ and did not adversely affect hepatic parasite burdens. Furthermore, the proparasitic properties of WT exosomes were not species specific because BALB/c mice exposed to Leishmania major exosomes showed increased Th2 polarization and exacerbation of disease in response to infection with L. major. These findings demonstrate that leishmania exosomes are predominantly immunosuppressive. Moreover, to our knowledge, this is the first evidence to suggest that changes in the protein cargo of exosomes may influence the impact of these vesicles on myeloid cell function.

  8. Distinct repertoires of microRNAs present in mouse astrocytes compared to astrocyte-secreted exosomes.

    Science.gov (United States)

    Jovičić, Ana; Gitler, Aaron D

    2017-01-01

    Astrocytes are the most abundant cell type in the central nervous system (CNS) and secrete various factors that regulate neuron development, function and connectivity. microRNAs (miRNAs) are small regulatory RNAs involved in posttranslational gene regulation. Recent findings showed that miRNAs are exchanged between cells via nanovesicles called exosomes. In this study, we sought to define which miRNAs are contained within exosomes secreted by astrocytes. We also explored whether astroglial miRNA secretion via exosomes is perturbed in a mouse model of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease where astrocytes play a crucial role in driving disease progression. By isolating and profiling the expression of miRNAs from primary mouse astrocytes and from the exosomes that astrocytes secrete, we compared miRNA expression in the cells and secreted vesicles. We established that miRNA expression profiles of astrocytes and their exosomes are vastly different. In addition, we determined that exosomal miRNA expression in astrocytes is not significantly perturbed in a mouse model of ALS. Astrocytes secrete numerous miRNAs via exosomes and miRNA species contained in exosomes are considerably different from miRNAs detectable in astrocytes, suggesting the existence of a mechanism to select certain miRNAs for inclusion or exclusion from exosomes. The exosomal miRNA profiling dataset we have generated will provide a resource to aid in the investigation of this selection mechanism. Finally, the miRNA expression profile in astrocyte-secreted exosomes is not perturbed by expression of mutant SOD1-G93A.

  9. Macrophage-derived exosomes induce inflammatory factors in endothelial cells under hypertensive conditions.

    Science.gov (United States)

    Osada-Oka, Mayuko; Shiota, Masayuki; Izumi, Yasukatsu; Nishiyama, Masaki; Tanaka, Masako; Yamaguchi, Takehiro; Sakurai, Emi; Miura, Katsuyuki; Iwao, Hiroshi

    2017-04-01

    Hypertension is one of the most important cardiovascular risk factors and results in macrophage infiltration of blood vessels. However, how macrophages coordinate inflammatory responses with endothelial cells (ECs) remains unclear. In this study, we investigated whether exosomes upregulate the expression of inflammatory factors in ECs under hypertensive conditions. Hypertension was induced in rats by continuous infusion of angiotensin II (Ang II). Exosomes were purified from rat serum by density gradient and ultracentrifugation and used to stimulate human coronary artery ECs (HCAECs). Moreover, the interactions between HCAECs and exosomes from human THP-1-derived macrophages were analyzed. Administration of Ang II enhanced the expression of CD68, a macrophage marker, in rat hearts, suggesting enhanced infiltration of macrophages. In addition, the expression of intracellular adhesion molecule-1 (ICAM1) and plasminogen activator inhibitor-1 (PAI-1), a proinflammatory factor, was increased in hypertensive rat hearts compared with control rats. CD68 protein expression and an increase in the expression of some exosome markers were detected in exosomes from hypertensive rat serum. Moreover, the exosomes upregulated the expression levels of ICAM1 and PAI-1 in HCAECs. The level of miR-17, a negative regulator of ICAM1 expression, was markedly decreased in exosomes from hypertensive rat serum compared with exosomes from control rats. Interestingly, Ang II-stimulated THP-1-derived exosomes also enhanced the expression of ICAM1 and PAI-1 and contained reduced levels of miR-17 compared with exosomes from unstimulated cells. These results suggest that inflammation of ECs under hypertensive conditions is caused, at least in part, by macrophage-derived exosomes.

  10. Can urinary exosomes act as treatment response markers in prostate cancer?

    Directory of Open Access Journals (Sweden)

    Tabi Zsuzsanna

    2009-01-01

    Full Text Available Abstract Background Recently, nanometer sized vesicles (termed exosomes have been described as a component of urine. Such vesicles may be a useful non-invasive source of markers in renal disease. Their utility as a source of markers in urological cancer remains unstudied. Our aim in this study was to investigate the feasibility and value of analysing urinary exosomes in prostate cancer patients undergoing standard therapy. Methods Ten patients (with locally advanced PCa provided spot urine specimens at three time points during standard therapy. Patients received 3–6 months neoadjuvant androgen deprivation therapy prior to radical radiotherapy, comprising a single phase delivering 55 Gy in 20 fractions to the prostate and 44 Gy in 20 fractions to the pelvic nodes. Patients were continued on adjuvant ADT according to clinical need. Exosomes were purified, and the phenotype compared to exosomes isolated from the prostate cancer cell line LNcaP. A control group of 10 healthy donors was included. Serum PSA was used as a surrogate treatment response marker. Exosomes present in urine were quantified, and expression of prostate markers (PSA and PSMA and tumour-associated marker 5T4 was examined. Results The quantity and quality of exosomes present in urine was highly variable, even though we handled all materials freshly and used methods optimized for obtaining highly pure exosomes. There was approx 2-fold decrease in urinary exosome content following 12 weeks ADT, but this was not sustained during radiotherapy. Nevertheless, PSA and PSMA were present in 20 of 24 PCa specimens, and not detected in healthy donor specimens. There was a clear treatment-related decrease in exosomal prostate markers in 1 (of 8 patient. Conclusion Evaluating urinary-exosomes remains difficult, given the variability of exosomes in urine specimens. Nevertheless, this approach holds promise as a non-invasive source of multiple markers of malignancy that could provide

  11. Proteomic analysis of exosomes from nasopharyngeal carcinoma cell identifies intercellular transfer of angiogenic proteins

    KAUST Repository

    Chan, Yuk-kit

    2015-04-01

    Exosomes, a group of secreted extracellular nanovesicles containing genetic materials and signaling molecules, play a critical role in intercellular communication. During tumorigenesis, exosomes have been demonstrated to promote tumor angiogenesis and metastasis while their biological functions in nasopharyngeal carcinoma (NPC) are poorly understood. In this study, we focused on the role of NPC-derived exosomes on angiogenesis. Exosomes derived from the NPC C666-1 cells and immortalized nasopharyngeal epithelial cells (NP69 and NP460) were isolated using ultracentrifugation. The molecular profile and biophysical characteristics of exosomes were verified by Western blotting, sucrose density gradient, and electron microscopy. We showed that the C666-1 exosomes (10 and 20 μg/ml) could significantly increase the tubulogenesis, migration and invasion of human umbilical vein endothelial cells (HUVECs) in a dose-dependent manner. Subsequently, an iTRAQ-based quantitative proteomics was used to identify the differentially expressed proteins in C666-1 exosomes. Among the 640 identified proteins, 51 and 89 proteins were considered as up- and down-regulated (≥ 1.5-fold variations) in C666-1 exosomes compared to the normal counterparts, respectively. As expected, pro-angiogenic proteins including intercellular adhesion molecule-1 (ICAM-1) and CD44 variant isoform 5 (CD44v5) are among the up-regulated proteins, whereas angio-suppressive protein, thrombospondin-1 (TSP-1) was down-regulated in C666-1 exosomes. Further confocal microscopic study and Western blotting clearly demonstrated that the alteration of ICAM-1, and TSP-1 expressions in recipient HUVECs are due to internalization of exosomes. Taken together, these data strongly indicated the critical roles of identified angiogenic proteins in the involvement of exosomes-induced angiogenesis, which could potentially be developed as therapeutic targets in future. This article is protected by copyright. All rights reserved.

  12. The role of exosomes in peripheral nerve regeneration

    Directory of Open Access Journals (Sweden)

    Rosanna C Ching

    2015-01-01

    Full Text Available Peripheral nerve injuries remain problematic to treat, with poor functional recovery commonly observed. Injuries resulting in a nerve gap create specific difficulties for axonal regeneration. Approaches to address these difficulties include autologous nerve grafts (which are currently the gold standard treatment and synthetic conduits, with the latter option being able to be impregnated with Schwann cells or stem cells which provide an appropriate micro-environment for neuronal regeneration to occur. Transplanting stem cells, however, infers additional risk of malignant transformation as well as manufacturing difficulties and ethical concerns, and the use of autologous nerve grafts and Schwann cells requires the sacrifice of a functioning nerve. A new approach utilizing exosomes, secreted extracellular vesicles, could avoid these complications. In this review, we summarize the current literature on exosomes, and suggest how they could help to improve axonal regeneration following peripheral nerve injury.

  13. Exosomes in Development and Therapy of Malignant Mesothelioma

    Science.gov (United States)

    2015-09-01

    exosomes. B. Dynamic light scattering (DLS) showing the size of the exosomes. 6 Histone H4 OS= Homo sapiens GN=HIST1H4A PE=1 SV=2 H4_HUMAN Keratin, type I...cytoskeletal 9 OS= Homo sapiens GN=KRT9 PE=1 SV=3 K1C9_HUMAN Isoform 2 of Clathrin heavy chain 1 OS= Homo sapiens GN=CLTC sp|Q00610-2|CLH1_HUMAN (+1...Clathrin heavy chain 2 OS= Homo sapiens GN=CLTCL1 PE=1 SV=2 sp|P53675|CLH2_HUMAN Cluster of Histone H2A type 1-B/E OS= Homo sapiens GN=HIST1H2AB PE=1 SV

  14. Tumour exosomes display different differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness

    DEFF Research Database (Denmark)

    Whitehead, Bradley Joseph; Wu, Linping; Hvam, Michael Lykke

    2015-01-01

    Background : Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell-derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells a......-malignant cell line exosomes, which may explain the observed increased endothelial monolayer disruption and transendothelial transport of these vesicles....

  15. Brain Gene Expression Signatures From Cerebrospinal Fluid Exosome RNA Profiling

    Science.gov (United States)

    Zanello, S. B.; Stevens, B.; Calvillo, E.; Tang, R.; Gutierrez Flores, B.; Hu, L.; Skog, J.; Bershad, E.

    2016-01-01

    While the Visual Impairment and Intracranial Pressure (VIIP) syndrome observations have focused on ocular symptoms, spaceflight has been also associated with a number of other performance and neurologic signs, such as headaches, cognitive changes, vertigo, nausea, sleep/circadian disruption and mood alterations, which, albeit likely multifactorial, can also result from elevation of intracranial pressure (ICP). We therefore hypothesize that these various symptoms are caused by disturbances in the neurophysiology of the brain structures and are correlated with molecular markers in the cerebrospinal fluid (CSF) as indicators of neurophysiological changes. Exosomes are 30-200 nm microvesicles shed into all biofluids, including blood, urine, and CSF, carrying a highly rich source of intact protein and RNA cargo. Exosomes have been identified in human CSF, and their proteome and RNA pool is a potential new reservoir for biomarker discovery in neurological disorders. The purpose of this study is to investigate changes in brain gene expression via exosome analysis in patients suffering from ICP elevation of varied severity (idiopathic intracranial hypertension -IIH), a condition which shares some of the neuroophthalmological features of VIIP, as a first step toward obtaining evidence suggesting that cognitive function and ICP levels can be correlated with biomarkers in the CSF. Our preliminary work, reported last year, validated the exosomal technology applicable to CSF analysis and demonstrated that it was possible to obtain gene expression evidence of inflammation processes in traumatic brain injury patients. We are now recruiting patients with suspected IIH requiring lumbar puncture at Baylor College of Medicine. Both CSF (5 ml) and human plasma (10 ml) are being collected in order to compare the pattern of differentially expressed genes observed in CSF and in blood. Since blood is much more accessible than CSF, we would like to determine whether plasma biomarkers for

  16. Preventing Prostate Cancer Metastasis by Targeting Exosome Secretion

    Science.gov (United States)

    2015-12-01

    metastatic). To avoid contamination by exosomes from the fetal bovine serum (FBS) supplement that is part of mammalian cell growth media , we...She, M.; Song, H.; Parker, R. Decapping activators in Saccharomyces cerevisiae act by multiple mechanisms. Mol. Cell 2010, 39, 773–783. 19. Su, H... growth . Testing this hypothesis has direct relevance towards an understanding of the often lethal complications of prostate cancer, manifested in

  17. KRS: A cut away from release in exosomes.

    Science.gov (United States)

    Rabouille, Catherine

    2017-07-03

    Cancer cells often trigger an inflammatory process, which in some cases may be driven by the presence of lysyl-tRNA synthetase (KRS) in the medium. Kim et al. (2017. J. Cell Biol. https://doi.org/10.1083/jcb.201605118) now demonstrate that cleavage of the KRS by caspase-8 inside cells triggers its interaction with syntenin and its release in inflammatory exosomes. © 2017 Rabouille.

  18. Exosomes: a potential key target in cardio-renal syndrome

    Directory of Open Access Journals (Sweden)

    Laura eGonzalez-Calero

    2014-10-01

    Full Text Available Exosomes have proven roles in regulating immune response, antigen presentation, RNA and protein transfer, and cell–cell (organ–organ interaction/signaling. These microvesicles can be considered a mechanism of non-classical secretion of proteins, and they represent a sub-proteome, thus assisting in the difficult task of biomarker discovery in a biological fluid as urine, plasma or serum. A potential role of exosomes in the cardio-renal syndrome is currently underexplored. Cardiovascular disease (CVD continues to be the leading cause of morbidity and mortality worldwide and, particularly, rates of cardiovascular events and death consistently increase as kidney function worsens. In other words, chronic kidney disease acts as a risk multiplier. Unfortunately, the relationship between markers of cardiovascular risk in kidney pathology often differs from that in the general population. Efforts in the search for novel action mechanisms simultaneously operating in both pathologies are thus of maximum interest.This article focuses to the role of exosomes in cardiovascular and renal diseases, in the search for novel key targets of interaction between heart and kidneys.

  19. The nuclear exosome is active and important during budding yeast meiosis.

    Directory of Open Access Journals (Sweden)

    Stephen Frenk

    Full Text Available Nuclear RNA degradation pathways are highly conserved across eukaryotes and play important roles in RNA quality control. Key substrates for exosomal degradation include aberrant functional RNAs and cryptic unstable transcripts (CUTs. It has recently been reported that the nuclear exosome is inactivated during meiosis in budding yeast through degradation of the subunit Rrp6, leading to the stabilisation of a subset of meiotic unannotated transcripts (MUTs of unknown function. We have analysed the activity of the nuclear exosome during meiosis by deletion of TRF4, which encodes a key component of the exosome targeting complex TRAMP. We find that TRAMP mutants produce high levels of CUTs during meiosis that are undetectable in wild-type cells, showing that the nuclear exosome remains functional for CUT degradation, and we further report that the meiotic exosome complex contains Rrp6. Indeed Rrp6 over-expression is insufficient to suppress MUT transcripts, showing that the reduced amount of Rrp6 in meiotic cells does not directly cause MUT accumulation. Lack of TRAMP activity stabilises ∼ 1600 CUTs in meiotic cells, which occupy 40% of the binding capacity of the nuclear cap binding complex (CBC. CBC mutants display defects in the formation of meiotic double strand breaks (DSBs, and we see similar defects in TRAMP mutants, suggesting that a key function of the nuclear exosome is to prevent saturation of the CBC complex by CUTs. Together, our results show that the nuclear exosome remains active in meiosis and has an important role in facilitating meiotic recombination.

  20. Multiple myeloma exosomes establish a favourable bone marrow microenvironment with enhanced angiogenesis and immunosuppression.

    Science.gov (United States)

    Wang, Jinheng; De Veirman, Kim; Faict, Sylvia; Frassanito, Maria Antonia; Ribatti, Domenico; Vacca, Angelo; Menu, Eline

    2016-06-01

    Multiple myeloma (MM) pathogenesis and progression largely rely on the cells and extracellular factors in the bone marrow (BM) microenvironment. Compelling studies have identified tumour exosomes as key regulators in the maintenance and education of the BM microenvironment by targeting stromal cells, immune cells, and vascular cells. However, the role of MM exosomes in the modification of the BM microenvironment and MM progression remains unclear. Here, we explored the functions of MM exosomes in angiogenesis and immunosuppression in vitro and in vivo. Murine MM exosomes carrying multiple angiogenesis-related proteins enhanced angiogenesis and directly promoted endothelial cell growth. Several pathways such as signal transducer and activator of transcription 3 (STAT3), c-Jun N-terminal kinase, and p53 were modulated by the exosomes in endothelial and BM stromal cells. These exosomes promoted the growth of myeloid-derived suppressor cells (MDSCs) in naive mice through activation of the STAT3 pathway and changed their subsets to similar phenotypes to those seen in MM-bearing mice. Moreover, MM exosomes up-regulated inducible nitric oxide synthase and enhanced the immunosuppressive capacity of BM MDSCs in vivo. Our data show that MM exosomes modulate the BM microenvironment through enhancement of angiogenesis and immunosuppression, which will further facilitate MM progression. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

  1. Tumor Microenvironment Modulation via Gold Nanoparticles Targeting Malicious Exosomes: Implications for Cancer Diagnostics and Therapy.

    Science.gov (United States)

    Roma-Rodrigues, Catarina; Raposo, Luís R; Cabral, Rita; Paradinha, Fabiana; Baptista, Pedro V; Fernandes, Alexandra R

    2017-01-14

    Exosomes are nanovesicles formed in the endosomal pathway with an important role in paracrine and autocrine cell communication. Exosomes secreted by cancer cells, malicious exosomes, have important roles in tumor microenvironment maturation and cancer progression. The knowledge of the role of exosomes in tumorigenesis prompted a new era in cancer diagnostics and therapy, taking advantage of the use of circulating exosomes as tumor biomarkers due to their stability in body fluids and targeting malignant exosomes' release and/or uptake to inhibit or delay tumor development. In recent years, nanotechnology has paved the way for the development of a plethora of new diagnostic and therapeutic platforms, fostering theranostics. The unique physical and chemical properties of gold nanoparticles (AuNPs) make them suitable vehicles to pursuit this goal. AuNPs' properties such as ease of synthesis with the desired shape and size, high surface:volume ratio, and the possibility of engineering their surface as desired, potentiate AuNPs' role in nanotheranostics, allowing the use of the same formulation for exosome detection and restraining the effect of malicious exosomes in cancer progression.

  2. Exosomes from B cells and Dendritic cells: mechanisms of formation, secretion and targeting

    NARCIS (Netherlands)

    Buschow, S.I.

    2006-01-01

    Many cell types, including dendritic cells (DC) and B cells, secrete small vesicles called exosomes. Exosomes from immune cells are thought to have immuno-regulatory functions but their precise role remains unresolved. The aim of the studies presented in this thesis was to get more insight into the

  3. Characterization of Uptake and Internalization of Exosomes by Bladder Cancer Cells

    Directory of Open Access Journals (Sweden)

    Carrie A. Franzen

    2014-01-01

    Full Text Available Bladder tumors represent a special therapeutic challenge as they have a high recurrence rate requiring repeated interventions and may progress to invasive or metastatic disease. Exosomes carry proteins implicated in bladder cancer progression and have been implicated in bladder cancer cell survival. Here, we characterized exosome uptake and internalization by human bladder cancer cells using Amnis ImageStreamX, an image cytometer. Exosomes were isolated by ultracentrifugation from bladder cancer culture conditioned supernatant, labeled with PKH-26, and analyzed on the ImageStreamX with an internal standard added to determine concentration. Exosomes were cocultured with bladder cancer cells and analyzed for internalization. Using the IDEAS software, we determined exosome uptake based on the number of PKH-26+ spots and overall PKH-26 fluorescence intensity. Using unlabeled beads of a known concentration and size, we were able to determine concentrations of exosomes isolated from bladder cancer cells. We measured exosome uptake by recipient bladder cancer cells, and we demonstrated that uptake is dose and time dependent. Finally, we found that uptake is active and specific, which can be partially blocked by heparin treatment. The characterization of cellular uptake and internalization by bladder cancer cells may shed light on the role of exosomes on bladder cancer recurrence and progression.

  4. Motile hepatocellular carcinoma cells preferentially secret sugar metabolism regulatory proteins via exosomes.

    Science.gov (United States)

    Zhang, Jing; Lu, Shaohua; Zhou, Ye; Meng, Kun; Chen, Zhipeng; Cui, Yizhi; Shi, Yunfeng; Wang, Tong; He, Qing-Yu

    2017-07-01

    Exosomes are deliverers of critically functional proteins, capable of transforming target cells in numerous cancers, including hepatocellular carcinoma (HCC). We hypothesize that the motility of HCC cells can be featured by comparative proteome of exosomes. Hence, we performed the super-SILAC-based MS analysis on the exosomes secreted by three human HCC cell lines, including the non-motile Hep3B cell, and the motile 97H and LM3 cells. More than 1400 exosomal proteins were confidently quantified in each MS analysis with highly biological reproducibility. We justified that 469 and 443 exosomal proteins represented differentially expressed proteins (DEPs) in the 97H/Hep3B and LM3/Hep3B comparisons, respectively. These DEPs focused on sugar metabolism-centric canonical pathways per ingenuity pathway analysis, which was consistent with the gene ontology analysis on biological process enrichment. These pathways included glycolysis I, gluconeogenesis I and pentose phosphate pathways; and the DEPs enriched in these pathways could form a tightly connected network. By analyzing the relative abundance of proteins and translating mRNAs, we found significantly positive correlation between exosomes and cells. The involved exosomal proteins were again focusing on sugar metabolism. In conclusion, motile HCC cells tend to preferentially export more sugar metabolism-associated proteins via exosomes that differentiate them from non-motile HCC cells. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. Acquisition of new tumor cell properties by MSC-derived exosomes.

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    Yang, Yuanyuan; Bucan, Vesna; Baehre, Heike; von der Ohe, Juliane; Otte, Anna; Hass, Ralf

    2015-07-01

    Interaction between multi-functional mesenchymal stroma/stem cells (MSC) and human tumor cells involves the exchange of biological material via extracellular vesicles including exosomes. Protein analysis of MSC-derived exosomes demonstrated the presence of MMP-2 and MSC-specific markers including CD90 and ecto-5'-nucleotidase (CD73). Incubation of tumor cells with these membranous particles revealed a rapid uptake of MSC-released microvesicles whereby breast cancer cells incorporated ~19% and SCCOHT-1 cells representing a rare type of small cell ovarian cancer assimilated ~28% of available exosomes within 24 h. This interaction was accompanied by functional alterations of tumor cell properties during integration of exosomal content from MSC. Indeed, exosome-associated MMP-2 exhibited functional enzyme activity and MCF-7 breast cancer cells with undetectable MMP-2 protein acquired expression of this enzyme and corresponding gelatinase functionality after stimulation with MSC-derived exosomes. Similar effects were observed in SCCOHT-1 cells during culture in the presence of MSC-derived exosomes which enabled new metabolic activities in this tumor cell type. Together, these findings demonstrated that the internalization of MSC-derived exosomes was associated with the acquisition of new tumor cell properties by altering cellular functionalities and providing the capability to re-organize the tumor microenvironment.

  6. Shikonin Inhibits the Proliferation of Human Breast Cancer Cells by Reducing Tumor-Derived Exosomes

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    Yao Wei

    2016-06-01

    Full Text Available Shikonin is a naphthoquinone isolated from the traditional Chinese medicine Lithospermum. It has been used in the treatment of various tumors. However, the effects of shikonin on such diseases have not been fully elucidated. In the present study, we detected the exosome release of a breast cancer cell line (MCF-7 with shikonin treatment and found a positive relationship between the level of secreted exosomes and cell proliferation. We next analyzed miRNA profiles in MCF-7 cells and exosomes and found that some miRNAs are specifically sorted and abundant in exosomes. Knockdown of the most abundant miRNAs in exosomes and the MCF-7 proliferation assay showed that miR-128 in exosomes negatively regulates the level of Bax in MCF-7 recipient cells and inhibits cell proliferation. These results show that shikonin inhibits the proliferation of MCF-7 cells through reducing tumor-derived exosomal miR-128. The current study suggests that shikonin suppresses MCF-7 growth by the inhibition of exosome release.

  7. Exosomal proteins as prognostic biomarkers in non-small cell lung cancer

    DEFF Research Database (Denmark)

    Paulsen, Birgitte Sandfeld; Aggerholm-Pedersen, N; Bæk, R

    2016-01-01

    BACKGROUND: Use of exosomes as biomarkers in non-small cell lung cancer (NSCLC) is an intriguing approach in the liquid-biopsy era. Exosomes are nano-sized vesicles with membrane-bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection...

  8. Exosome-SIRPα, a CD47 blockade increases cancer cell phagocytosis.

    Science.gov (United States)

    Koh, Eunee; Lee, Eun Jung; Nam, Gi-Hoon; Hong, Yeonsun; Cho, Eunji; Yang, Yoosoo; Kim, In-San

    2017-03-01

    CD47, a "don't eat me" signal, is over-expressed on the surface of most tumors that interacts with signal regulatory protein α (SIRPα) on phagocytic cells. By engaging SIRPα, CD47 limits the ability of macrophages to engulf tumor cells, which acts as a major phagocytic barrier. In this study, we developed an exosome-based immune checkpoint blockade that antagonizes the interaction between CD47 and SIRPα. These exosomes harboring SIRPα variants (SIRPα-exosomes) were sufficient to induce remarkably augmented tumor phagocytosis, lead to prime effective anti-tumor T cell response. Given that clustering of native CD47 provides a high binding avidity to ligate dimerized SIRPα on macrophage, nature-derived exosomes could be appreciable platform to antagonize CD47. Disruption of CD47-SIRPα interaction by SIRPα-exosomes leads to an increase in cells being engulfed by macrophages and a concomitant inhibition of tumor growth in tumor-bearing mice. Moreover, SIRPα-exosomes therapy promotes an intensive T cell infiltration in syngeneic mouse models of cancer, raising the possibility of CD47-targeted therapies to unleash both an innate and adaptive anti-tumor response. Note that very small amount of exosomal SIRPα proteins could effectively lead to phagocytic elimination of tumor cells both in vitro and in vivo. Our results suggest that superlative exosome-based platform has broad potential to maximize the therapeutic efficacy of membrane-associated protein therapeutics. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. Exosomal Proteins as Diagnostic Biomarkers in Lung Cancer

    DEFF Research Database (Denmark)

    Sandfeld-Paulsen, Birgitte; Jakobsen, Kristine Raaby; Bæk, Rikke

    2016-01-01

    histological subtypes in patients. METHODS: Plasma was isolated from 581 patients (431 with lung cancer and 150 controls). The extracellular vesicle array was used to phenotype exosomes. The extracellular vesicle array contained 49 antibodies for capturing exosomes. Subsequently, a cocktail of biotin...

  10. Effects of subtoxic concentrations of TiO2 and ZnO nanoparticles on human lymphocytes, dendritic cells and exosome production.

    Science.gov (United States)

    Andersson-Willman, Britta; Gehrmann, Ulf; Cansu, Zekiye; Buerki-Thurnherr, Tina; Krug, Harald F; Gabrielsson, Susanne; Scheynius, Annika

    2012-10-01

    Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, but the knowledge of their possible interactions with the immune system is very limited. Our aims were to investigate if commercially available TiO(2) and ZnO nanoparticles may affect different human immune cells and their production of exosomes, nano-sized vesicles that have a role in cell to cell communication. We found that the TiO(2) or ZnO nanoparticles at concentrations from 1 to 100μg/mL did not affect the viability of primary human peripheral blood mononuclear cells (PBMC). In contrast, monocyte-derived dendritic cells (MDDC) reacted with a dose dependent increase in cell death and caspase activity to ZnO but not to TiO(2) nanoparticles. Non-toxic exposure, 10μg/mL, to TiO(2) and ZnO nanoparticles did not significantly alter the phenotype of MDDC. Interestingly, ZnO but not TiO(2) nanoparticles induced a down regulation of FcγRIII (CD16) expression on NK-cells in the PBMC population, suggesting that subtoxic concentrations of ZnO nanoparticles might have an effect on FcγR-mediated immune responses. The phenotype and size of exosomes produced by PBMC or MDDC exposed to the nanoparticles were similar to that of exosomes harvested from control cultures. TiO(2) or ZnO nanoparticles could not be detected within or associated to exosomes as analyzed with TEM. We conclude that TiO(2) and ZnO nanoparticles differently affect immune cells and that evaluations of nanoparticles should be performed even at subtoxic concentrations on different primary human immune cells when investigating potential effects on immune functions. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Drosophila TG-A transglutaminase is secreted via an unconventional Golgi-independent mechanism involving exosomes and two types of fatty acylations.

    Science.gov (United States)

    Shibata, Toshio; Hadano, Jinki; Kawasaki, Daichi; Dong, Xiaoqing; Kawabata, Shun-Ichiro

    2017-06-23

    Transglutaminases (TGs) play essential intracellular and extracellular roles by covalently cross-linking many proteins. Drosophila TG is encoded by one gene and has two alternative splicing-derived isoforms, TG-A and TG-B, which contain distinct N-terminal 46- and 38-amino acid sequences, respectively. The TGs identified to date do not have a typical endoplasmic reticulum (ER)-signal peptide, and the molecular mechanisms of their secretion under physiologic conditions are unclear. Immunocytochemistry revealed that TG-A localizes to multivesicular-like structures, whereas TG-B localizes to the cytosol. We also found that TG-A, but not TG-B, was modified concomitantly by N-myristoylation and S-palmitoylation, and N-myristoylation was a pre-requisite for S-palmitoylation. Moreover, TG-A, but not TG-B, was secreted in response to calcium signaling induced by Ca(2+) ionophores and uracil, a pathogenic bacteria-derived substance. Brefeldin A and monensin, inhibitors of the ER/Golgi-mediated conventional pathway, did not suppress TG-A secretion, whereas inhibition of S-palmitoylation by 2-bromopalmitate blocked TG-A secretion. Ultracentrifugation, electron microscopy analyses, and treatments with inhibitors of multivesicular body formation revealed that TG-A was secreted via exosomes together with co-transfected mammalian CD63, an exosomal marker, and the secreted TG-A was taken up by other cells. The 8-residue N-terminal fragment of TG-A containing the fatty acylation sites was both necessary and sufficient for the exosome-dependent secretion of TG-A. In conclusion, TG-A is secreted through an unconventional ER/Golgi-independent pathway involving two types of fatty acylations and exosomes. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  12. MSC exosome as a cell-free MSC therapy for cartilage regeneration: Implications for osteoarthritis treatment.

    Science.gov (United States)

    Toh, Wei Seong; Lai, Ruenn Chai; Hui, James Hoi Po; Lim, Sai Kiang

    2017-07-01

    Mesenchymal stem cell (MSC) therapies have demonstrated efficacy in cartilage repair in animal and clinical studies. The efficacy of MSC-based therapies which was previously predicated on the chondrogenic potential of MSC is increasingly attributed to the paracrine secretion, particularly exosomes. Exosomes are thought to function primarily as intercellular communication vehicles to transfer bioactive lipids, nucleic acids (mRNAs and microRNAs) and proteins between cells to elicit biological responses in recipient cells. For MSC exosomes, many of these biological responses translated to a therapeutic outcome in injured or diseased cells. Here, we review the current understanding of MSC exosomes, discuss the possible mechanisms of action in cartilage repair within the context of the widely reported immunomodulatory and regenerative potency of MSC exosomes, and provide new perspectives for development of an off-the-shelf and cell-free MSC therapy for treatment of cartilage injuries and osteoarthritis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. Placental exosomes and pre-eclampsia: Maternal circulating levels in normal pregnancies and, early and late onset pre-eclamptic pregnancies.

    Science.gov (United States)

    Pillay, Preenan; Maharaj, Niren; Moodley, Jagidesa; Mackraj, Irene

    2016-10-01

    Exosomes are a subtype of extracellular vesicle (20-130 nm) released by biological cells under normal and pathological conditions. Although there have been reports of circulating exosomes in normal pregnancy, the relevance of placental-derived exosomes in normal and abnormal pregnancies still needs to be elucidated. The aim of this study was to quantify total and placental-derived exosomes in maternal plasma from normal (N), early onset- and late onset-preeclampsia (PE). Plasma samples were obtained from pregnant women in the third trimester, for the isolation of exosomes by differential ultracentrifugation. Total exosomes were quantified using nanoparticle tracking analysis and immuno-reactive exosomal CD63 quantification. Placental-derived exosomes were quantified using placental alkaline phosphatase (PLAP) as a specific marker. The contribution of placental-derived exosomes to total exosomes in maternal plasma was determined by the ratio of PLAP(+) exosomes to CD63(+) exosomes. The concentration of total exosomes significantly increased in early onset-PE and late onset-PE compared to N (≤33 weeks) and N (≥34 weeks). The relative concentration of placental-derived exosomes significantly increased in early onset-PE but decreased in late onset-PE compared to N. The ratio of PLAP(+) exosomes to total number of exosomes significantly decreased in early onset-PE and late onset-PE. A positive correlation between total and placental-derived exosomes were obtained in N (≤33 weeks: Pearson's r = 0.60, ≥34 weeks: Pearson's r = 0.67) and early onset-PE (Pearson's r = 0.51, p exosomes to total exosomes in maternal circulation suggests a possible pathophysiological role of placental-derived exosomes in pre-eclampsia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Morphological and molecular features of oral fluid-derived exosomes: oral cancer patients versus healthy individuals.

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    Zlotogorski-Hurvitz, Ayelet; Dayan, Dan; Chaushu, Gavriel; Salo, Tuula; Vered, Marilena

    2016-01-01

    Oral cancer (OC) patients are at high risk to develop recurrent disease or secondary primary cancers with no available biomarkers to detect these events until a visible lesion is readily present and diagnosed by biopsy. Exosomes secreted by cancer cells are involved in tumor growth, invasion and metastasis. We aimed to determine morphological and molecular differences between oral fluid (OF)-derived exosomes of OC patients and those isolated from healthy individuals (HI). OF from OC patients (n = 36) and HI (n = 25) was initially assessed by nanoparticle tracking analysis (NTA). Following ultracentrifugation, exosomal pellets of OC patients and HI were morphologically examined by transmission electron microscopy and atomic force microscopy (AFM). Enzyme-linked immunosorbent assay (ELISA) and western blotting (WB) were used to analyze the expression of exosomal markers--CD9, CD81 and CD63. NTA showed that OC samples of OF had a significantly higher concentration of nanoparticles/ml (p = 0.01) and modal nanoparticle size (p = 0.002) compared to HI. The difference in size was structurally highlighted by AFM three-dimensional images applied on exosomal pellets. ELISA and WB showed differential expression of exosomal markers in OC exosomes compared to HI: lower expression of CD81 and CD9 in contrast to a higher expression of CD63 (~53 kDa). OF-derived exosomes from OC patients differ both morphologically and molecularly from exosomes present in HI. This study is a baseline that provides a starting point for finding exosomal biomarkers for early detection of malignant changes in high-risk patients without overt clinical signs/lesions.

  15. Asymmetric RNA Distribution among Cells and Their Secreted Exosomes: Biomedical Meaning and Considerations on Diagnostic Applications

    Directory of Open Access Journals (Sweden)

    Marco Ragusa

    2017-10-01

    Full Text Available Over the past few years, exosomes and their RNA cargo have been extensively studied because of the fascinating biological roles they play in cell-to-cell communication, including the signal exchange among cancer, stromal, and immune cells, leading to modifications of tumor microenvironment. RNAs, especially miRNAs, stored within exosomes, seem to be among the main determinants of such signaling: their sorting into exosomes appears to be cell-specific and related to cellular physiopathology. Accordingly, the identification of exosomal miRNAs in body fluids from pathological patients has become one of the most promising activity in the field of biomarker discovery. Several analyses on the qualitative and quantitative distribution of RNAs between cells and their secreted exosomes have given rise to questions on whether and how accurately exosomal RNAs would represent the transcriptomic snapshot of the physiological and pathological status of secreting cells. Although the exact molecular mechanisms of sorting remain quite elusive, many papers have reported an evident asymmetric quantitative distribution of RNAs between source cells and their exosomes. This phenomenon could depend both on passive and active sorting mechanisms related to: (a RNA turnover; (b maintaining the cytoplasmic miRNA:target equilibrium; (c removal of RNAs not critical or even detrimental for normal or diseased cells. These observations represent very critical issues in the exploitation of exosomal miRNAs as cancer biomarkers. In this review, we will discuss how much the exosomal and corresponding donor cell transcriptomes match each other, to better understand the actual reliability of exosomal RNA molecules as pathological biomarkers reflecting a diseased status of the cells.

  16. Exosomes as biomimetic tools for stem cell differentiation: Applications in dental pulp tissue regeneration.

    Science.gov (United States)

    Huang, Chun-Chieh; Narayanan, Raghuvaran; Alapati, Satish; Ravindran, Sriram

    2016-12-01

    Achieving and maintaining safe and reliable lineage specific differentiation of stem cells is important for clinical translation of tissue engineering strategies. In an effort to circumvent the multitude of problems arising from the usage of growth factors and growth factor delivery systems, we have explored the use of exosomes as biomimetic tools to induce stem cell differentiation. Working on the hypothesis that cell-type specific exosomes can trigger lineage-specific differentiation of stem cells, we have evaluated the potential of exosomes derived from dental pulp cells cultured on under growth and odontogenic differentiation conditions to induce odontogenic differentiation of naïve human dental pulp stem cells (DPSCs) and human bone marrow derived stromal cells (HMSCs) in vitro and in vivo. Results indicate that the exosomes can bind to matrix proteins such as type I collagen and fibronectin enabling them to be tethered to biomaterials. The exosomes are endocytosed by both DPSCs and HMSCs in a dose-dependent and saturable manner via the caveolar endocytic mechanism and trigger the P38 mitogen activated protein kinase (MAPK) pathway. In addition, the exosomes also trigger the increased expression of genes required for odontogenic differentiation. When tested in vivo in a tooth root slice model with DPSCs, the exosomes triggered regeneration of dental pulp-like tissue. However, our results indicate that exosomes isolated under odontogenic conditions are better inducers of stem cell differentiation and tissue regeneration. Overall, our results highlight the potential exosomes as biomimetic tools to induce lineage specific differentiation of stem cells. Our results also show the importance of considering the source and state of exosome donor cells before a choice is made for therapeutic applications. Copyright © 2016 Elsevier Ltd. All rights reserved.

  17. Effect on Intermittent Hypoxia on Plasma Exosomal Micro RNA Signature and Endothelial Function in Healthy Adults.

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    Khalyfa, Abdelnaby; Zhang, Chunling; Khalyfa, Ahamed A; Foster, Glen E; Beaudin, Andrew E; Andrade, Jorge; Hanly, Patrick J; Poulin, Marc J; Gozal, David

    2016-12-01

    Intermittent hypoxia (IH) is associated with increased risk of cardiovascular disease. Exosomes are secreted by most cell types and released in biological fluids, including plasma, and play a role in modifying the functional phenotype of target cells. Using an experimental human model of IH, we investigated potential exosome-derived biomarkers of IH-induced vascular dysfunction. Ten male volunteers were exposed to room air (D0), IH (6 h/day) for 4 days (D4) and allowed to recover for 4 days (D8). Circulating plasma exosomes were isolated and incubated with human endothelial monolayer cultures for impedance measurements and RNA extracted and processed with messenger RNA (mRNA) arrays to identify gene targets. In addition, immunofluorescent assessments of endothelial nitric oxide synthase (eNOS) mRNA expression, ICAM-1 cellular distribution were conducted. Plasma exosomal micro RNAs (miRNAs) were profiled. D4 exosomes, primarily from endothelial sources, disrupted impedance levels compared to D0 and D8. ICAM-1 expression was markedly upregulated in endothelial cells exposed to D4 exosomes along with significant reductions in eNOS expression. Microarray approaches identified a restricted and further validated signature of exosomal miRNAs in D4 exosomes, and mRNA arrays revealed putative endothelial gene target pathways. In humans, intermittent hypoxia alters exosome cargo in the circulation which promotes increased permeability and dysfunction of endothelial cells in vitro. A select number of circulating exosomal miRNAs may play important roles in the cardiovascular dysfunction associated with OSA by targeting specific effector pathways.

  18. ATL-derived exosomes modulate mesenchymal stem cells: potential role in leukemia progression.

    Science.gov (United States)

    El-Saghir, Jamal; Nassar, Farah; Tawil, Nadim; El-Sabban, Marwan

    2016-10-19

    Exosomes are membrane nano-vesicles secreted by a multitude of cells that harbor biological constituents such as proteins, lipids, mRNA and microRNA. Exosomes can potentially transfer their cargo to other cells, implicating them in many patho-physiological processes. Mesenchymal stem cells (MSCs), residents of the bone marrow and metastatic niches, potentially interact with cancer cells and/or their derived exosomes. In this study, we investigated whether exosomes derived from adult T-cell leukemia/lymphoma (ATL) cells act as intercellular messengers delivering leukemia-related genes that modulate the properties of human MSCs in favor of leukemia. We hypothesized that the cargo of ATL-derived exosomes is transferred to MSCs and alter their functional behavior to support the establishment of the appropriate microenvironment for leukemia. We showed that both ATL cells (C81 and HuT-102) and patient-derived cells released Tax-containing exosomes. The cargo of HuT-102-derived exosomes consisted of miR-21, miR-155 and vascular endothelial growth factor. We demonstrated that HuT-102-derived exosomes not only deliver Tax to recipient MSCs, but also induce NF-κB activation leading to a change in cellular morphology, increase in proliferation and the induction of gene expression of migration and angiogenic markers. This study demonstrates that ATL-derived exosomes deliver Tax and other leukemia-related genes to MSCs and alter their properties to presumably create a more conducive milieu for leukemia. These findings highlight the contribution of leukemia-derived exosomes in cellular transformation and their potential value as biomarkers and targets in therapeutic strategies.

  19. Identifying mRNA, MicroRNA and Protein Profiles of Melanoma Exosomes

    Science.gov (United States)

    Chen, Yinlu; Taylor, Douglas D.; Rai, Shesh N.; Waigel, Sabine; Zacharias, Wolfgang; Hao, Hongying; McMasters, Kelly M.

    2012-01-01

    Background Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells, and in various disease conditions. Tumor exosomes contain intact and functional mRNAs, small RNAs (including miRNAs), and proteins that can alter the cellular environment to favor tumor growth. Molecular profiling may increase our understanding of the role of exosomes in melanoma progression and may lead to discovery of useful biomarkers. Methodology/Principal Findings In the present study, we used mRNA array profiling to identify thousands of exosomal mRNAs associated with melanoma progression and metastasis. Similarly, miRNA array profiling identified specific miRNAs, such as hsa-miR-31, -185, and -34b, involved in melanoma invasion. We also used proteomic analysis and discovered differentially expressed melanoma exosomal proteins, including HAPLN1, GRP78, syntenin-1, annexin A1, and annexin A2. Importantly, normal melanocytes acquired invasion ability through molecules transported in melanoma cell-derived exosomes. Conclusions/Significance Our results indicate that melanoma-derived exosomes have unique gene expression signatures, miRNA and proteomics profiles compared to exosomes from normal melanocytes. To the best of our knowledge, this is the first in-depth screening of the whole transcriptome/miRNome/proteome expression in melanoma exosomes. These results provide a starting point for future more in-depth studies of tumor-derived melanoma exosomes, which will aid our understanding of melanoma biogenesis and new drug-targets that may be translated into clinical applications, or as non-invasive biomarkers for melanoma. PMID:23056502

  20. Dromedary milk exosomes as mammary transcriptome nano-vehicle: Their isolation, vesicular and phospholipidomic characterizatio

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    Aya M. Yassin

    2016-09-01

    Full Text Available Exosomes are extracellular nanovesicles that play a role in cellular trafficking and communication. Camel milk exosomes might carry the potential of recovery of several illnesses that coins the dromedary milk. This study shows for the first time their isolation and fine characterization. The differential ultracentrifugation was used for their isolation. Their recovery from dromedary milk during different lactation periods was evaluated. The vesicular characterization and stability testing of the recovered exosome were examined by transmission electron microscopy (TEM. The proteome footprinting was resolved by gel electrophoresis prior to their specific protein biomarker analysis. The immunoblotting of their specific protein biomarker TSG101 unexpectedly revealed a truncated 35 KDa protein specific for dromedary milk exosome rather than the previously reported 43 KDa mammalian one. The reversed-phase HPLC screening of their phospholipid makeup was compared with that of cattle milk exosomes at different lactation periods. Since dromedary milk exosomes reflect their mammary transcriptome outcome, further assessment of their content of αs1casein, αs2casein β-casein κ-casein mRNAs parallel with a constitutive glyceraldehyde dehydrogenase (GAPD gene was performed using real-time PCR. The TEM scanning indicated that dromedary milk exosomes are freeze-stress unstable homogeneous with average size of 30 nm. There was no significant difference in expression level of different casein genes in mid lactation period in dromedary milk exosomes over late lactation period. The phospholipidomic survey proved that phosphatidylcholine is the major candidate of the examined phospholipids in dromedary milk exosomes. The obtained data give novel interpretation about the content of camel milk exosomes with possible insight for use as potentially-safe nano carrier.

  1. Exosomes derived from tumor cells genetically modified to express Mycobacterium tuberculosis antigen: a novel vaccine for cancer therapy.

    Science.gov (United States)

    Koyama, Yoshiyuki; Ito, Tomoko; Hasegawa, Aya; Eriguchi, Masazumi; Inaba, Toshio; Ushigusa, Takahiro; Sugiura, Kikuya

    2016-11-01

    To examine the potential of exosomes derived from the tumor cells, which had been genetically modified to express a Mycobacterium tuberculosis antigen, as a cancer vaccine aimed at overcoming the weak immunogenicity of tumor antigens. We transfected B16 melanoma cells with a plasmid encoding the M. tuberculosis antigen, early secretory antigenic target-6 (ESAT-6). The secreted exosomes bearing both tumor-associated antigens and the pathogenic antigen (or their epitopes) were collected. When the exosomes were injected into foot pads of mice, they significantly (p exosomes significantly suppressed (p exosomes derived from the non-transfected B16 cells showed no effect on tumor growth, although both exosomes should have similar tumor antigens. Exosomes bearing both tumor antigens and the M. tuberculosis antigen (or their epitopes) have a high potential as a candidate for cancer vaccine to overcome the immune escape by tumor cells.

  2. Toward Exosome-Based Therapeutics: Isolation, Heterogeneity, and Fit-for-Purpose Potency

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    Gareth R. Willis

    2017-10-01

    Full Text Available Exosomes are defined as submicron (30–150 nm, lipid bilayer-enclosed extracellular vesicles (EVs, specifically generated by the late endosomal compartment through fusion of multivesicular bodies with the plasma membrane. Produced by almost all cells, exosomes were originally considered to represent just a mechanism for jettisoning unwanted cellular moieties. Although this may be a major function in most cells, evolution has recruited the endosomal membrane-sorting pathway to duties beyond mere garbage disposal, one of the most notable examples being its cooption by retroviruses for the generation of Trojan virions. It is, therefore, tempting to speculate that certain cell types have evolved an exosome subclass active in intracellular communication. We term this EV subclass “signalosomes” and define them as exosomes that are produced by the “signaling” cells upon specific physiological or environmental cues and harbor cargo capable of modulating the programming of recipient cells. Our recent studies have established that signalosomes released by mesenchymal stem/stromal cells (MSCs represent the main vector of MSC immunomodulation and therapeutic action in animal models of lung disease. The efficacy of MSC-exosome treatments in a number of preclinical models of cardiovascular and pulmonary disease supports the promise of application of exosome-based therapeutics across a wide range of pathologies within the near future. However, the full realization of exosome therapeutic potential has been hampered by the absence of standardization in EV isolation, and procedures for purification of signalosomes from the main exosome population. This is mainly due to immature methodologies for exosome isolation and characterization and our incomplete understanding of the specific characteristics and molecular composition of signalosomes. In addition, difficulties in defining metrics for potency of exosome preparations and the challenges of industrial

  3. MicroRNA profiling in kidney disease: Plasma versus plasma-derived exosomes.

    Science.gov (United States)

    Xie, Jeffrey X; Fan, Xiaoming; Drummond, Christopher A; Majumder, Reetam; Xie, Yanmei; Chen, Tian; Liu, Lijun; Haller, Steven T; Brewster, Pamela S; Dworkin, Lance D; Cooper, Christopher J; Tian, Jiang

    2017-09-05

    Liquid biopsies have advanced rapidly in recent years for use in diagnostic and prognostic applications. One important aspect of this advancement is the growth in our understanding of microRNA (miRNA) biology. The measurement of miRNAs packaged within exosomes, which are constantly released into the blood stream, may reflect pathological changes within the body. The current study performed miRNA profiling using plasma and plasma-derived exosome samples from two animal models of kidney disease, the 5/6th partial nephrectomy (PNx) and two-kidney-one-clip (2K1C) models. The RT-qPCR-based profiling results revealed that the overall miRNA expression level was much higher in plasma than in plasma-derived exosomes. With 200μl of either plasma or exosomes derived from the same volume of plasma, 629 out of 665 total miRNAs analyzed were detectable in plasma samples from sham-operated rats, while only 403 were detectable in exosomes with a cutoff value set at 35cycles. Moreover, the average miRNA expression level in plasma was about 16-fold higher than that in exosomes. We also found a select subset of miRNAs that were enriched within exosomes. The number of detectable miRNAs from plasma-derived exosomes was increased in rats subjected to PNx or 2K1C surgery compared to sham-operated animals. Importantly, we found that the changes of individual miRNAs measured in plasma had very poor concordance with that measured in plasma-derived exosomes in both animal models, suggesting that miRNAs in plasma and plasma-derived exosomes are differentially regulated in these disease conditions. Interestingly, PNx and 2K1C surgeries induced similar changes in miRNA expression, implying that common pathways were activated in these two disease models. Pathway analyses using DIANA-miRPath v3.0 showed that significantly changed exosomal miRNAs were associated with extracellular matrix (ECM) receptor interaction and mucin type-O-glycan synthesis pathways, which are related with tissue fibrosis

  4. Amnion-Epithelial-Cell-Derived Exosomes Demonstrate Physiologic State of Cell under Oxidative Stress.

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    Samantha Sheller

    Full Text Available At term, the signals of fetal maturity and feto-placental tissue aging prompt uterine readiness for delivery by transitioning quiescent myometrium to an active stage. It is still unclear how the signals reach the distant myometrium. Exosomes are a specific type of extracellular vesicle (EVs that transport molecular signals between cells, and are released from a wide range of cells, including the maternal and fetal cells. In this study, we hypothesize that i exosomes act as carriers of signals in utero-placental compartments and ii exosomes reflect the physiologic status of the origin cells. The primary aims of this study were to determine exosomal contents in exosomes derived from primary amnion epithelial cells (AEC. We also determined the effect of oxidative stress on AEC derived exosomal cargo contents. AEC were isolated from amniotic membrane obtained from normal, term, not in labor placentae at delivery, and culture under standard conditions. Oxidative stress was induced using cigarette smoke extract for 48 hours. AEC-conditioned media were collected and exosomes isolated by differential centrifugations. Both growth conditions (normal and oxidative stress induced produced cup shaped exosomes of around 50 nm, expressed exosomes enriched markers, such as CD9, CD63, CD81 and HSC70, embryonic stem cell marker Nanog, and contained similar amounts of cell free AEC DNA. Using confocal microscopy, the colocalization of histone (H 3, heat shock protein (HSP 70 and activated form of pro-senescence and term parturition associated marker p38 mitogen activated protein kinase (MAPK (P-p38 MAPK co-localized with exosome enrich marker CD9. HSP70 and P-p38 MAPK were significantly higher in exosomes from AEC grown under oxidative stress conditions than standard conditions (p<0.05. Finally, mass spectrometry and bioinformatics analysis identified 221 different proteins involved in immunomodulatory response and cell-to-cell communication. This study determined

  5. MSCs-Derived Exosomes and Neuroinflammation, Neurogenesis and Therapy of Traumatic Brain Injury

    Science.gov (United States)

    Yang, Yongxiang; Ye, Yuqin; Su, Xinhong; He, Jun; Bai, Wei; He, Xiaosheng

    2017-01-01

    Exosomes are endosomal origin membrane-enclosed small vesicles (30–100 nm) that contain various molecular constituents including proteins, lipids, mRNAs and microRNAs. Accumulating studies demonstrated that exosomes initiated and regulated neuroinflammation, modified neurogenic niches and neurogenesis, and were even of potential significance in treating some neurological diseases. These tiny extracellular vesicles (EVs) can derive from some kinds of multipotent cells such as mesenchymal stem cells (MSCs) that have been confirmed to be a potentially promising therapy for traumatic brain injury (TBI) in experimental models and in preclinical studies. Nevertheless, subsequent studies demonstrated that the predominant mechanisms of MSCs’s contributions to brain tissue repairment and functional recovery after TBI were not the cell replacement effects but likely the secretion-based paracrine effects produced by EVs such as MSCs-derived exosomes. These nanosized exosomes derived from MSCs cannot proliferate, are easier to preserve and transfer and have lower immunogenicity, compared with transplanted exogenous MSCs. These reports revealed that MSCs-derived exosomes might promise to be a new and valuable therapeutic strategy for TBI than MSCs themselves. However, the concrete mechanisms involved in the positive effects induced by MSCs-derived exosomes in TBI are still ambiguous. In this review, we intend to explore the potential effects of MSCs-derived exosomes on neuroinflammation and neurogenesis in TBI and, especially, on therapy. PMID:28293177

  6. Tumor Microenvironment Modulation via Gold Nanoparticles Targeting Malicious Exosomes: Implications for Cancer Diagnostics and Therapy

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    Catarina Roma-Rodrigues

    2017-01-01

    Full Text Available Exosomes are nanovesicles formed in the endosomal pathway with an important role in paracrine and autocrine cell communication. Exosomes secreted by cancer cells, malicious exosomes, have important roles in tumor microenvironment maturation and cancer progression. The knowledge of the role of exosomes in tumorigenesis prompted a new era in cancer diagnostics and therapy, taking advantage of the use of circulating exosomes as tumor biomarkers due to their stability in body fluids and targeting malignant exosomes’ release and/or uptake to inhibit or delay tumor development. In recent years, nanotechnology has paved the way for the development of a plethora of new diagnostic and therapeutic platforms, fostering theranostics. The unique physical and chemical properties of gold nanoparticles (AuNPs make them suitable vehicles to pursuit this goal. AuNPs’ properties such as ease of synthesis with the desired shape and size, high surface:volume ratio, and the possibility of engineering their surface as desired, potentiate AuNPs’ role in nanotheranostics, allowing the use of the same formulation for exosome detection and restraining the effect of malicious exosomes in cancer progression.

  7. Isolation and characterization of exosomes from blood of patients with mastopathy and breast cancer

    Science.gov (United States)

    Tamkovich, S. N.; Somov, A. K.; Karpukhina, K. V.; Grigor'eva, A. E.; Yunusova, N. V.; Stakheeva, M. N.; Voytsitskiy, V. E.; Kondakova, I. V.; Laktionov, P. P.

    2017-09-01

    The exosomes containing tumor-specific protein represent a valuable source of material for the non-invasive diagnosis of malignant neoplasms. The exosomes from plasma and cell-associated exosomes from blood of healthy women, the patients with mastopathy and breast cancer patients were studied with the nanoparticle tracking analysis, transmission electron microscopy, flow cytometry and protein profiling. The exposure of CD63, CD24, CD9, and CD81 demonstrates isolation of mainly exosomes. A major part of exosomes in the blood of healthy women, patients with mastopathy and breast cancer patients is shown to be associated with the surface of blood cells. The concentration of the exosomes in the plasma of the breast cancer patients is higher than in the healthy women's plasma. The proteins ranged from 10 to 250 kDa were found in the exosomes by gradient SDS PAAGE; 8 regions of electrophoregram differ between healthy and illness patients in the expression level and number of proteins. These proteins are planned to be identified by MALDI TOF and studied as potential breast cancer markers.

  8. Stem Cell-Derived Exosomes: A Potential Alternative Therapeutic Agent in Orthopaedics

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    John Burke

    2016-01-01

    Full Text Available Within the field of regenerative medicine, many have sought to use stem cells as a promising way to heal human tissue; however, in the past few years, exosomes (packaged vesicles released from cells have shown more exciting promise. Specifically, stem cell-derived exosomes have demonstrated great ability to provide therapeutical benefits. Exosomal products can include miRNA, other genetic products, proteins, and various factors. They are released from cells in a paracrine fashion in order to combat local cellular stress. Because of this, there are vast benefits that medicine can obtain from stem cell-derived exosomes. If exosomes could be extracted from stem cells in an efficient manner and packaged with particular regenerative products, then diseases such as rheumatoid arthritis, osteoarthritis, bone fractures, and other maladies could be treated with cell-free regenerative medicine via exosomes. Many advances must be made to get to this point, and the following review highlights the current advances of stem cell-derived exosomes with particular attention to regenerative medicine in orthopaedics.

  9. Exosomes and other extracellular vesicles in neural cells and neurodegenerative diseases.

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    Janas, Anna M; Sapoń, Karolina; Janas, Teresa; Stowell, Michael H B; Janas, Tadeusz

    2016-06-01

    The function of human nervous system is critically dependent on proper interneuronal communication. Exosomes and other extracellular vesicles are emerging as a novel form of information exchange within the nervous system. Intraluminal vesicles within multivesicular bodies (MVBs) can be transported in neural cells anterogradely or retrogradely in order to be released into the extracellular space as exosomes. RNA loading into exosomes can be either via an interaction between RNA and the raft-like region of the MVB limiting membrane, or via an interaction between an RNA-binding protein-RNA complex with this raft-like region. Outflow of exosomes from neural cells and inflow of exosomes into neural cells presumably take place on a continuous basis. Exosomes can play both neuro-protective and neuro-toxic roles. In this review, we characterize the role of exosomes and microvesicles in normal nervous system function, and summarize evidence for defective signaling of these vesicles in disease pathogenesis of some neurodegenerative diseases. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Stem cell-derived exosomes: A promising strategy for fracture healing.

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    Hao, Zi-Chen; Lu, Jun; Wang, Shan-Zheng; Wu, Hao; Zhang, Yun-Tong; Xu, Shuo-Gui

    2017-10-01

    To describe the biological characteristics of exosomes and to summarize the current status of stem cell-derived exosomes on fracture healing. Meanwhile, future challenges, limitations and perspectives are also discussed. Search and analyze the related articles in pubmed database through the multi-combination of keywords like "stem cells","exosomes","bone regeneration" and "fracture healing". Stem cell-derived exosome therapy for fracture healing has been enjoying popularity and is drawing increasing attention. This strategy helps to promote proliferation and migration of cells, as well as osteogenesis and angiogenesis, in the process of bone formation. Although the exact mechanisms remain elusive, exosomal miRNAs seem to play vital roles. Future studies are required to solve multiple problems before clinical application, including comprehensive and thorough understanding of exosomes, the exact roles of exosomes in regulating bone formation, and the optimal source, dose and frequency of treatment, as well as technical and safety issues. Moreover, studies based on fracture models of large animals are could offer guidance and are in demand. © 2017 John Wiley & Sons Ltd.

  11. Fractionation of Exosomes and DNA using Size-Based Separation at the Nanoscale

    Science.gov (United States)

    Wunsch, Benjamin; Smith, Joshua; Wang, Chao; Gifford, Stacey; Brink, Markus; Bruce, Robert; Solovitzky, Gustavo; Austin, Robert; Astier, Yann

    Exosomes, a key target of ``liquid biopsies'', are nano-vesicles found in nearly all biological fluids. Exosomes are secreted by eukaryotic and prokaryotic cells alike, and contain information about their originating cells, including surface proteins, cytoplasmic proteins, and nucleic acids. One challenge in studying exosome morphology is the difficulty of sorting exosomes by size and surface markers. Common separation techniques for exosomes include ultracentrifugation and ultrafiltration, for preparation of large volume samples, but these techniques often show contamination and significant heterogeneity between preparations. To date, deterministic lateral displacement (DLD) pillar arrays in silicon have proven an efficient technology to sort, separate, and enrich micron-scale particles including human parasites, eukaryotic cells, blood cells, and circulating tumor cells in blood; however, the DLD technology has never been translated to the true nanoscale, where it could function on bio-colloids such as exosomes. We have fabricated nanoscale DLD (nanoDLD) arrays capable of rapidly sorting colloids down to 20 nm in continuous flow, and demonstrated size sorting of individual exosome vesicles and dsDNA polymers, opening the potential for on-chip biomolecule separation and diagnosti

  12. Exosome in Tumour Microenvironment: Overview of the Crosstalk between Normal and Cancer Cells

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    Catarina Roma-Rodrigues

    2014-01-01

    Full Text Available Cancer development is a multistep process in which exosomes play important roles. Exosomes are small vesicles formed in vesicular bodies in the endosomal network. The major role of exosomes seems to be the transport of bioactive molecules between cells. Depending on the cell of origin, exosomes are implicated in the regulation of several cellular events, with phenotypic consequences in recipient cells. Cancer derived exosomes (CCEs are important players in the formation of the tumour microenvironment by (i enabling the escape of tumour cells to immunological system and help initiating the inflammatory response; (ii acting in the differentiation of fibroblasts and mesenchymal cells into myofibroblasts; (iii triggering the angiogenic process; and (iv enhancing the metastatic evolution of the tumour by promoting epithelial to mesenchymal transformation of tumour cells and by preparing the tumour niche in the new anatomical location. Since the finding that exosomes content resembles that of the cell of origin, they may be regarded as suitable biomarkers for cancer diagnosis, allowing for diagnosis and prognosis via a minimal invasive procedure. Exosome involvement in cancer may open new avenues regarding therapeutics, such as vectors for targeted drug delivery.

  13. Exosomal transfer of proteins and RNAs at synapses in the nervous system

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    Smalheiser Neil R

    2007-11-01

    Full Text Available Abstract Background Many cell types have been reported to secrete small vesicles called exosomes, that are derived from multivesicular bodies and that can also form from endocytic-like lipid raft domains of the plasma membrane. Secretory exosomes contain a characteristic composition of proteins, and a recent report indicates that mast cell exosomes harbor a variety of mRNAs and microRNAs as well. Exosomes express cell recognition molecules on their surface that facilitate their selective targeting and uptake into recipient cells. Results In this review, I suggest that exosomal secretion of proteins and RNAs may be a fundamental mode of communication within the nervous system, supplementing the known mechanisms of anterograde and retrograde signaling across synapses. In one specific scenario, exosomes are proposed to bud from the lipid raft region of the postsynaptic membrane adjacent to the postsynaptic density, in a manner that is stimulated by stimuli that elicit long-term potentiation. The exosomes would then transfer newly synthesized synaptic proteins (such as CAM kinase II alpha and synaptic RNAs to the presynaptic terminal, where they would contribute to synaptic plasticity. Conclusion The model is consistent with the known cellular and molecular features of synaptic neurobiology and makes a number of predictions that can be tested in vitro and in vivo. Open peer review Reviewed by Etienne Joly, Gaspar Jekely, Juergen Brosius and Eugene Koonin. For the full reviews, please go to the Reviewers' comments section.

  14. ExoCarta: A Web-Based Compendium of Exosomal Cargo.

    Science.gov (United States)

    Keerthikumar, Shivakumar; Chisanga, David; Ariyaratne, Dinuka; Al Saffar, Haidar; Anand, Sushma; Zhao, Kening; Samuel, Monisha; Pathan, Mohashin; Jois, Markandeya; Chilamkurti, Naveen; Gangoda, Lahiru; Mathivanan, Suresh

    2016-02-22

    Exosomes are membranous vesicles that are released by a variety of cells into the extracellular microenvironment and are implicated in intercellular communication. As exosomes contain RNA, proteins and lipids, there is a significant interest in characterizing the molecular cargo of exosomes. Here, we describe ExoCarta (http://www.exocarta.org), a manually curated Web-based compendium of exosomal proteins, RNAs and lipids. Since its inception, the database has been highly accessed (>54,000 visitors from 135 countries). The current version of ExoCarta hosts 41,860 proteins, >7540 RNA and 1116 lipid molecules from more than 286 exosomal studies annotated with International Society for Extracellular Vesicles minimal experimental requirements for definition of extracellular vesicles. Besides, ExoCarta features dynamic protein-protein interaction networks and biological pathways of exosomal proteins. Users can download most often identified exosomal proteins based on the number of studies. The downloaded files can further be imported directly into FunRich (http://www.funrich.org) tool for additional functional enrichment and interaction network analysis. Copyright © 2016 Elsevier Ltd. All rights reserved.

  15. Surface functionalized exosomes as targeted drug delivery vehicles for cerebral ischemia therapy.

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    Tian, Tian; Zhang, Hui-Xin; He, Chun-Peng; Fan, Song; Zhu, Yan-Liang; Qi, Cui; Huang, Ning-Ping; Xiao, Zhong-Dang; Lu, Zu-Hong; Tannous, Bakhos A; Gao, Jun

    2018-01-01

    The safe and effective delivery of drugs is a major obstacle in the treatment of ischemic stroke. Exosomes hold great promise as an endogenous drug delivery nanosystem for the treatment of cerebral ischemia given their unique properties, including low immunogenicity, innate stability, high delivery efficiency, and ability to cross the blood-brain barrier (BBB). However, exosome insufficient targeting capability limits their clinical applications. In this study, the c(RGDyK) peptide has been conjugated to the exosome surface by an easy, rapid, and bio-orthogonal chemistry. In the transient middle cerebral artery occlusion (MCAO) mice model, The engineered c(RGDyK)-conjugated exosomes (cRGD-Exo) target the lesion region of the ischemic brain after intravenous administration. Furthermore, curcumin has been loaded onto the cRGD-Exo, and administration of these exosomes has resulted in a strong suppression of the inflammatory response and cellular apoptosis in the lesion region. The results suggest a targeting delivery vehicle for ischemic brain based on exosomes and provide a strategy for the rapid and large-scale production of functionalized exosomes. Copyright © 2017 Elsevier Ltd. All rights reserved.

  16. Structure determination of an 11-subunit exosome in complex with RNA by molecular replacement

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    Makino, Debora Lika, E-mail: dmakino@biochem.mpg.de; Conti, Elena [Max Planck Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried (Germany)

    2013-11-01

    The crystallographic steps towards the structure determination of a complete eukaryotic exosome complex bound to RNA are presented. Phasing of this 11-protein subunit complex was carried out via molecular replacement. The RNA exosome is an evolutionarily conserved multi-protein complex involved in the 3′ degradation of a variety of RNA transcripts. In the nucleus, the exosome participates in the maturation of structured RNAs, in the surveillance of pre-mRNAs and in the decay of a variety of noncoding transcripts. In the cytoplasm, the exosome degrades mRNAs in constitutive and regulated turnover pathways. Several structures of subcomplexes of eukaryotic exosomes or related prokaryotic exosome-like complexes are known, but how the complete assembly is organized to fulfil processive RNA degradation has been unclear. An atomic snapshot of a Saccharomyces cerevisiae 420 kDa exosome complex bound to an RNA substrate in the pre-cleavage state of a hydrolytic reaction has been determined. Here, the crystallographic steps towards the structural elucidation, which was carried out by molecular replacement, are presented.

  17. Sialoglycoproteins and N-glycans from secreted exosomes of ovarian carcinoma cells.

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    Cristina Escrevente

    Full Text Available Exosomes consist of vesicles that are secreted by several human cells, including tumor cells and neurons, and they are found in several biological fluids. Exosomes have characteristic protein and lipid composition, however, the results concerning glycoprotein composition and glycosylation are scarce. Here, protein glycosylation of exosomes from ovarian carcinoma SKOV3 cells has been studied by lectin blotting, NP-HPLC analysis of 2-aminobenzamide labeled glycans and mass spectrometry. An abundant sialoglycoprotein was found enriched in exosomes and it was identified by peptide mass fingerprinting and immunoblot as the galectin-3-binding protein (LGALS3BP. Exosomes were found to contain predominantly complex glycans of the di-, tri-, and tetraantennary type with or without proximal fucose and also high mannose glycans. Diantennary glycans containing bisecting N-acetylglucosamine were also detected. This work provides detailed information about glycoprotein and N-glycan composition of exosomes from ovarian cancer cells, furthermore it opens novel perspectives to further explore the functional role of glycans in the biology of exosomes.

  18. CD44v6 Dependence of Premetastatic Niche Preparation by Exosomes

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    Thorsten Jung

    2009-10-01

    Full Text Available The metastasizing capacity of the rat pancreatic adenocarcinoma BSp73ASML (ASMLwt is strikingly reduced by a knockdown of CD44v4-v7 (ASMLkd. We used this model to analyze the role of the CD44 variant isoform (CD44v in (premetastatic niche formation. Intrafootpad injections of ASMLwt-, but not ASMLkd-conditioned medium (CM, strongly promote settlement of ASMLkd cells in lymph nodes and lung. Fractionation of CM revealed a contribution by a soluble matrix and exosomes, where the CD44v6-containing ASMLwt-soluble fraction can complement ASMLkd-exosomes, but not vice versa. This implies that exosomes are the final actors, are CD44v-independent, but require a soluble matrix, which depends on CD44v. Analyzing the composition revealed that only the ASMLwt-matrix contains c-Met and urokinase-type plasminogen activator receptor. In vitro, mostly ASMLwt-exosomes promote proliferation and induce gene expression in metastatic organ cells. However, in vivo corresponding changes in the (pre metastatic organ are only observed when both, exosomes plus the soluble matrix, are provided. Thus, neither CD44v nor exosomes alone suffice for (premetastatic niche formation. Instead, CD44v suffices for assembling a soluble matrix, which allows exosomes, independent of their origin from poorly or highly metastatic cells, to modulate (pre metastatic organ cells for tumor cell embedding and growth.

  19. Molecular lipid species in urinary exosomes as potential prostate cancer biomarkers.

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    Skotland, Tore; Ekroos, Kim; Kauhanen, Dimple; Simolin, Helena; Seierstad, Therese; Berge, Viktor; Sandvig, Kirsten; Llorente, Alicia

    2017-01-01

    Exosomes have recently appeared as a novel source of noninvasive cancer biomarkers, since these nanovesicles contain molecules from cancer cells and can be detected in biofluids. We have here investigated the potential use of lipids in urinary exosomes as prostate cancer biomarkers. A high-throughput mass spectrometry quantitative lipidomic analysis was performed to reveal the lipid composition of urinary exosomes in prostate cancer patients and healthy controls. Control samples were first analysed to characterise the lipidome of urinary exosomes and test the reproducibility of the method. In total, 107 lipid species were quantified in urinary exosomes. Several differences, for example, in cholesterol and phosphatidylcholine, were found between urinary exosomes and exosomes derived from cell lines, thus showing the importance of in vivo studies for biomarker analysis. The 36 most abundant lipid species in urinary exosomes were then quantified in 15 prostate cancer patients and 13 healthy controls. Interestingly, the levels of nine lipids species were found to be significantly different when the two groups were compared. The highest significance was shown for phosphatidylserine (PS) 18:1/18:1 and lactosylceramide (d18:1/16:0), the latter also showed the highest patient-to-control ratio. Furthermore, combinations of these lipid species and PS 18:0-18:2 distinguished the two groups with 93% sensitivity and 100% specificity. Finally, in agreement with the reported dysregulation of sphingolipid metabolism in cancer cells, alteration in specific sphingolipid lipid classes were observed. This study shows for the first time the potential use of exosomal lipid species in urine as prostate cancer biomarkers. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Tumor Cell Malignant Properties Are Enhanced by Circulating Exosomes in Sleep Apnea.

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    Almendros, Isaac; Khalyfa, Abdelnaby; Trzepizur, Wojciech; Gileles-Hillel, Alex; Huang, Lei; Akbarpour, Mahzad; Andrade, Jorge; Farré, Ramon; Gozal, David

    2016-11-01

    OSA is associated with increased cancer incidence and mortality. Exosomes are vesicles secreted by most cells. They are released into the bloodstream and play a role in tumor progression and metastasis. We evaluated whether the chronic intermittent hypoxia (IH) that characterizes OSA leads to release of tumor-promoting exosomes in the circulation. C57/B6 male mice were randomized to 6 weeks of IH or room air (RA). A subgroup was injected with TC1 lung carcinoma cells in the left flank after 2 weeks of IH. Exosomes from mouse plasma and from 10 adult human patients with OSA before and after treatment for 6 weeks were cocultured with mouse TC1 and human adenocarcinoma cells lines. Malignant tumor properties such as proliferation, migration, invasion, and endothelial monolayer disruption were assessed, as was micro-RNA (miRNA), exosomal content, and transcriptomic effects of exosomes on TC1 cells in vitro to identify target genes. Application of IH-induced exosomes from either IH-exposed tumor-bearing (IH+) or non-tumor-bearing (IH-) mice significantly promoted TC1 malignant properties. Similarly, before adherent treatment, exosomes from patients with OSA significantly enhanced proliferation and migration of human adenocarcinoma cells compared with after adherent treatment. Eleven distinct miRNAs emerged in IH-exposed mice, and their gene targets in TC1 cells were identified. Circulating exosomes released under IH conditions in vivo selectively enhance specific properties of lung tumor cell cultures. Thus, plasma exosomes participate in the increased tumor aggressiveness observed in patients with OSA. Copyright © 2016 American College of Chest Physicians. Published by Elsevier Inc. All rights reserved.

  1. Emerging roles of exosomes in normal and pathological conditions. New insights for diagnosis and therapeutic applications

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    Julieta eDe Toro

    2015-05-01

    Full Text Available From the time when they were first described in the 1970s by the group of Johnstone and Stahl, exosomes are a target of constant research. Exosomes belong to the family of nano-vesicles which are of great interest for their many functions and potential for diagnosis and therapy in multiples diseases. Exosomes originate from the intraluminal vesicles of late endosomal compartments named multivesicular bodies and the fusion of these late endosomes with the cell membrane result in the release of the vesicles into the extracellular compartment. Moreover, their generation can be induced by many factors including extracellular stimuli, such as microbial attack and other stress conditions. The primary role attributed to exosomes was the removal of unnecessary proteins from the cells. Now, several studies have demonstrated that exosomes are involved in cell-cell communication, even though their biological function is not completely clear.The participation of exosomes in cancer is the field of microvesicle research that has expanded more over the last years. Evidence proving that exosomes derived from tumor-pulsed dendritic cells, neoplastic cells and malignant effusions, are able to present antigens to T‐cells, has led to numerous studies using them as cell free cancer vaccines.Since exosomes derive from all cell types, they contain proteins, lipids and miRNA capable of regulating a variety of target genes. Much research is being conducted, which focuses on the employment of these vesicles as biomarkers in the diagnosis of cancer in addition to innovative biomarkers for diagnosis, prognosis and management of cardiovascular diseases. Interesting findings indicating the role of exosomes in the pathogenesis of several diseases have encouraged researchers to consider their therapeutic potential not only in oncology but also in the treatment of autoimmune syndromes and neurodegenerative disorders such as Alzheimer´s and Parkinson´s disease; in addition

  2. Tumour exosomes display differential mechanical and complement activation properties dependent on malignant state: implications in endothelial leakiness

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    Bradley Whitehead

    2015-12-01

    Full Text Available Background: Exosomes have been implicated in tumour progression and metastatic spread. Little is known of the effect of mechanical and innate immune interactions of malignant cell-derived exosomes on endothelial integrity, which may relate to increased extravasation of circulating tumour cells and, therefore, increased metastatic spread. Methods: Exosomes isolated from non-malignant immortalized HCV-29 and isogenic malignant non-metastatic T24 and malignant metastatic FL3 bladder cells were characterized by nanoparticle tracking analysis and quantitative nanomechanical mapping atomic force microscopy (QNM AFM to determine size and nanomechanical properties. Effect of HCV-29, T24 and FL3 exosomes on human umbilical vein endothelial cell (HUVEC monolayer integrity was determined by transendothelial electrical resistance (TEER measurements and transport was determined by flow cytometry. Complement activation studies in human serum of malignant and non-malignant cell-derived exosomes were performed. Results: FL3, T24 and HCV-29 cells produced exosomes at similar concentration per cell (6.64, 6.61 and 6.46×104 exosomes per cell for FL3, T24 and HCV-29 cells, respectively and of similar size (120.2 nm for FL3, 127.6 nm for T24 and 117.9 nm for HCV-29, respectively. T24 and FL3 cell-derived exosomes exhibited a markedly reduced stiffness, 95 MPa and 280 MPa, respectively, compared with 1,527 MPa with non-malignant HCV-29 cell-derived exosomes determined by QNM AFM. FL3 and T24 exosomes induced endothelial disruption as measured by a decrease in TEER in HUVEC monolayers, whereas no effect was observed for HCV-29 derived exosomes. FL3 and T24 exosomes traffic more readily (11.6 and 21.4% of applied exosomes, respectively across HUVEC monolayers than HCV-29 derived exosomes (7.2% of applied exosomes. Malignant cell-derived exosomes activated complement through calcium-sensitive pathways in a concentration-dependent manner. Conclusions: Malignant

  3. HucMSC Exosome-Delivered 14-3-3ζ Orchestrates Self-Control of the Wnt Response via Modulation of YAP During Cutaneous Regeneration.

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    Zhang, Bin; Shi, Yinghong; Gong, Aihua; Pan, Zhaoji; Shi, Hui; Yang, Huan; Fu, Hailong; Yan, Yongmin; Zhang, Xu; Wang, Mei; Zhu, Wei; Qian, Hui; Xu, Wenrong

    2016-10-01

    Numerous studies showed that mesenchymal stem cells derived exosome (MSC-Ex) markedly enhanced tissue regeneration, however, the issue of whether MSC-Ex could control stem cells expansion after a regenerative response to prevent tissue from overcrowding and dysplasia remains to be established. Herein, we found that human umbilical cord MSC (hucMSC)-exosomal14-3-3ζ mediated the binding of YAP and p-LATS by forming a complex to promote the phosphorylation of YAP, which orchestrate exosomal Wnt4 signal in cutaneous regeneration. First, we assessed deep second-degree burn rats treated with hucMSC-Ex and discovered that hucMSC-Ex promoting self-regulation of Wnt/β-catenin signaling at the remodeling phase of cutaneous regeneration. HucMSC-Ex restricted excessive skin cell expansion and collagen deposition at 4 weeks. Under high cell density conditions, hucMSC-Ex inhibited Wnt/β-catenin signaling through induction of YAP phosphorylation. Second, hucMSC-Ex proteomic analysis revealed that 14-3-3 proteins could be transported by exosome. Using gain- and loss-of-function studies, our results showed that hucMSC-exosomal 14-3-3ζ controlled YAP activities and phosphorylation at Ser127 site, and were required for the binding of YAP and p-LATS. Further studies revealed that 14-3-3ζ recruited YAP and p-LATS to form a complex under high cells density status and 14-3-3ζ other than YAP or p-LATS was the key regulatory molecule of this complex. These findings collectively indicate that hucMSC-Ex functions not only as an "accelerator" of the Wnt/β-catenin signal to repair damaged skin tissue but also as a "brake" of the signal by modulating YAP to orchestrate controlled cutaneous regeneration. Stem Cells 2016;34:2485-2500. © 2016 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Pres.

  4. Effects of subtoxic concentrations of TiO{sub 2} and ZnO nanoparticles on human lymphocytes, dendritic cells and exosome production

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    Andersson-Willman, Britta; Gehrmann, Ulf; Cansu, Zekiye [Translational Immunology Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm (Sweden); Buerki-Thurnherr, Tina; Krug, Harald F. [Laboratory for Materials — Biology Interactions, Swiss Federal Laboratories of Materials Testing and Research, St. Gallen (Switzerland); Gabrielsson, Susanne [Translational Immunology Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm (Sweden); Scheynius, Annika, E-mail: annika.scheynius@ki.se [Translational Immunology Unit, Department of Medicine Solna, Karolinska Institutet, Stockholm (Sweden)

    2012-10-01

    Metal oxide nanoparticles are widely used in the paint and coating industry as well as in cosmetics, but the knowledge of their possible interactions with the immune system is very limited. Our aims were to investigate if commercially available TiO{sub 2} and ZnO nanoparticles may affect different human immune cells and their production of exosomes, nano-sized vesicles that have a role in cell to cell communication. We found that the TiO{sub 2} or ZnO nanoparticles at concentrations from 1 to 100 μg/mL did not affect the viability of primary human peripheral blood mononuclear cells (PBMC). In contrast, monocyte-derived dendritic cells (MDDC) reacted with a dose dependent increase in cell death and caspase activity to ZnO but not to TiO{sub 2} nanoparticles. Non-toxic exposure, 10 μg/mL, to TiO{sub 2} and ZnO nanoparticles did not significantly alter the phenotype of MDDC. Interestingly, ZnO but not TiO{sub 2} nanoparticles induced a down regulation of FcγRIII (CD16) expression on NK-cells in the PBMC population, suggesting that subtoxic concentrations of ZnO nanoparticles might have an effect on FcγR-mediated immune responses. The phenotype and size of exosomes produced by PBMC or MDDC exposed to the nanoparticles were similar to that of exosomes harvested from control cultures. TiO{sub 2} or ZnO nanoparticles could not be detected within or associated to exosomes as analyzed with TEM. We conclude that TiO{sub 2} and ZnO nanoparticles differently affect immune cells and that evaluations of nanoparticles should be performed even at subtoxic concentrations on different primary human immune cells when investigating potential effects on immune functions. -- Highlights: ► ZnO nanoparticles induce cell death of MDDC but not of PBMC. ► ZnO nanoparticles induce caspase activation and DNA fragmentation in MDDC. ► TiO{sub 2} nanoparticles are taken up by MDDC but have no effect on their phenotype. ► ZnO nanoparticles induce a significant reduction of CD16

  5. Cell type-specific and common characteristics of exosomes derived from mouse cell lines: Yield, physicochemical properties, and pharmacokinetics.

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    Charoenviriyakul, Chonlada; Takahashi, Yuki; Morishita, Masaki; Matsumoto, Akihiro; Nishikawa, Makiya; Takakura, Yoshinobu

    2017-01-01

    Exosomes are small membrane vesicles secreted from cells and are expected to be used as drug delivery systems. Important characteristics of exosomes, such as yield, physicochemical properties, and pharmacokinetics, may be different among different cell types. However, there is limited information about the effect of cell type on these characteristics. In the present study, we evaluated these characteristics of exosomes derived from five different types of mouse cell lines: B16BL6 murine melanoma cells, C2C12 murine myoblast cells, NIH3T3 murine fibroblasts cells, MAEC murine aortic endothelial cells, and RAW264.7 murine macrophage-like cells. Exosomes were collected using a differential ultracentrifugation method. The exosomes collected from all the cell types were negatively charged globular vesicles with a diameter of approximately 100nm. C2C12 and RAW264.7 cells produced more exosomes than the other types of cells. The exosomes were labeled with a fusion protein of Gaussia luciferase and lactadherin to evaluate their pharmacokinetics. After intravenous injection into mice, all the exosomes rapidly disappeared from the systemic circulation and mainly distributed to the liver. In conclusion, the exosome yield was significantly different among the cell types, and all the exosomes evaluated in this study showed comparable physicochemical and pharmacokinetic properties. Copyright © 2016 Elsevier B.V. All rights reserved.

  6. Amyloid β-Exposed Human Astrocytes Overproduce Phospho-Tau and Overrelease It within Exosomes, Effects Suppressed by Calcilytic NPS 2143—Further Implications for Alzheimer's Therapy

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    Anna Chiarini

    2017-04-01

    Full Text Available The two main drivers of Alzheimer's disease (AD, amyloid-β (Aβ and hyperphosphorylated Tau (p-Tau oligomers, cooperatively accelerate AD progression, but a hot debate is still ongoing about which of the two appears first. Here we present preliminary evidence showing that Tau and p-Tau are expressed by untransformed cortical adult human astrocytes in culture and that exposure of such cells to an Aβ42 proxy, Aβ25−35, which binds the calcium-sensing receptor (CaSR and activates its signaling, significantly increases intracellular p-Tau levels, an effect CaSR antagonist (calcilytic NPS 2143 wholly hinders. The astrocytes also release both Tau and p-Tau by means of exosomes into the extracellular medium, an activity that could mediate p-Tau diffusion within the brain. Preliminary data also indicate that exosomal levels of p-Tau increase after Aβ25−35 exposure, but remain unchanged in cells pre-treated for 30-min with NPS 2143 before adding Aβ25−35. Thus, our previous and present findings raise the unifying prospect that Aβ•CaSR signaling plays a crucial role in AD development and progression by simultaneously activating (i the amyloidogenic processing of amyloid precursor holoprotein, whose upshot is a surplus production and secretion of Aβ42 oligomers, and (ii the GSK-3β-mediated increased production of p-Tau oligomers which are next released extracellularly inside exosomes. Therefore, as calcilytics suppress both effects on Aβ42 and p-Tau metabolic handling, these highly selective antagonists of pathological Aβ•CaSR signaling would effectively halt AD's progressive spread preserving patients' cognition and life quality.

  7. Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing.

    Science.gov (United States)

    Jenjaroenpun, Piroon; Kremenska, Yuliya; Nair, Vrundha M; Kremenskoy, Maksym; Joseph, Baby; Kurochkin, Igor V

    2013-01-01

    Exosomes are nanosized (30-100 nm) membrane vesicles secreted by most cell types. Exosomes have been found to contain various RNA species including miRNA, mRNA and long non-protein coding RNAs. A number of cancer cells produce elevated levels of exosomes. Because exosomes have been isolated from most body fluids they may provide a source for non-invasive cancer diagnostics. Transcriptome profiling that uses deep-sequencing technologies (RNA-Seq) offers enormous amount of data that can be used for biomarkers discovery, however, in case of exosomes this approach was applied only for the analysis of small RNAs. In this study, we utilized RNA-Seq technology to analyze RNAs present in microvesicles secreted by human breast cancer cell lines. Exosomes were isolated from the media conditioned by two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436. Exosomal RNA was profiled using the Ion Torrent semiconductor chip-based technology. Exosomes were found to contain various classes of RNA with the major class represented by fragmented ribosomal RNA (rRNA), in particular 28S and 18S rRNA subunits. Analysis of exosomal RNA content revealed that it reflects RNA content of the donor cells. Although exosomes produced by the two cancer cell lines shared most of the RNA species, there was a number of non-coding transcripts unique to MDA-MB-231 and MDA-MB-436 cells. This suggests that RNA analysis might distinguish exosomes produced by low metastatic breast cancer cell line (MDA-MB-436) from that produced by highly metastatic breast cancer cell line (MDA-MB-231). The analysis of gene ontologies (GOs) associated with the most abundant transcripts present in exosomes revealed significant enrichment in genes encoding proteins involved in translation and rRNA and ncRNA processing. These GO terms indicate most expressed genes for both, cellular and exosomal RNA. For the first time, using RNA-seq, we examined the transcriptomes of exosomes secreted by human breast cancer cells. We

  8. Characterization of RNA in exosomes secreted by human breast cancer cell lines using next-generation sequencing

    Directory of Open Access Journals (Sweden)

    Piroon Jenjaroenpun

    2013-11-01

    Full Text Available Exosomes are nanosized (30–100 nm membrane vesicles secreted by most cell types. Exosomes have been found to contain various RNA species including miRNA, mRNA and long non-protein coding RNAs. A number of cancer cells produce elevated levels of exosomes. Because exosomes have been isolated from most body fluids they may provide a source for non-invasive cancer diagnostics. Transcriptome profiling that uses deep-sequencing technologies (RNA-Seq offers enormous amount of data that can be used for biomarkers discovery, however, in case of exosomes this approach was applied only for the analysis of small RNAs. In this study, we utilized RNA-Seq technology to analyze RNAs present in microvesicles secreted by human breast cancer cell lines.Exosomes were isolated from the media conditioned by two human breast cancer cell lines, MDA-MB-231 and MDA-MB-436. Exosomal RNA was profiled using the Ion Torrent semiconductor chip-based technology. Exosomes were found to contain various classes of RNA with the major class represented by fragmented ribosomal RNA (rRNA, in particular 28S and 18S rRNA subunits. Analysis of exosomal RNA content revealed that it reflects RNA content of the donor cells. Although exosomes produced by the two cancer cell lines shared most of the RNA species, there was a number of non-coding transcripts unique to MDA-MB-231 and MDA-MB-436 cells. This suggests that RNA analysis might distinguish exosomes produced by low metastatic breast cancer cell line (MDA-MB-436 from that produced by highly metastatic breast cancer cell line (MDA-MB-231. The analysis of gene ontologies (GOs associated with the most abundant transcripts present in exosomes revealed significant enrichment in genes encoding proteins involved in translation and rRNA and ncRNA processing. These GO terms indicate most expressed genes for both, cellular and exosomal RNA.For the first time, using RNA-seq, we examined the transcriptomes of exosomes secreted by human breast

  9. Regulation of microvascularization in heart failure - an endothelial cell, non-coding RNAs and exosome liaison

    Directory of Open Access Journals (Sweden)

    Rio P. Juni

    2017-03-01

    Full Text Available Heart failure is a complex syndrome involving various pathophysiological processes. An increasing body of evidence shows that the myocardial microvasculature is essential for the homeostasis state and that a decompensated heart is associated with microvascular dysfunction as a result of impaired endothelial angiogenic capacity. The intercellular communication between endothelial cells and cardiomyocytes through various signaling molecules, such as vascular endothelial growth factor, nitric oxide, and non-coding RNAs is an important determinant of cardiac microvascular function. Non-coding RNAs are transported from endothelial cells to cardiomyocytes, and vice versa, regulating microvascular properties and angiogenic processes in the heart. Small-exocytosed vesicles, called exosomes, which are secreted by both cell types, can mediate this intercellular communication. The purpose of this review is to highlight the contribution of the microvasculature to proper heart function maintenance by focusing on the interaction between cardiac endothelial cells and myocytes with a specific emphasis on non-coding RNAs (ncRNAs in this form of cell-to-cell communication. Finally, the potential of ncRNAs as targets for angiogenesis therapy will also be discussed.

  10. The Induction of Protective Immunity against Experimental Eimeria tenella Infection using Serum Exosomes

    Science.gov (United States)

    Avian coccidiosis is caused by Eimeria, a unicellular, apicomplexan protist which primarily infects intestinal epithelia resulting in nutrition malabsorption and reduced growth of commercial poultry. Vaccination of chickens with exosomes isolated from antigen presenting cells and containing parasit...

  11. Exosomal Heat Shock Proteins as New Players in Tumour Cell-to-Cell Communication

    Directory of Open Access Journals (Sweden)

    Claudia Campanella

    2014-06-01

    Full Text Available Exosomes have recently been proposed as novel elements in the study of intercellular communication in normal and pathological conditions. The biomolecular composition of exosomes reflects the specialized functions of the original cells. Heat shock proteins (Hsps are a group of chaperone proteins with diverse biological roles. In recent years, many studies have focused on the extracellular roles played by Hsps that appear to be involved in cancer development and immune system stimulation. Hsps localized on the surface of exosomes, secreted by normal and tumour cells, could be key players in intercellular cross-talk, particularly during the course of different diseases, such as cancer. Exosomal Hsps offer significant opportunities for clinical applications, including their use as potential novel biomarkers for the diagnoses or prognoses of different diseases, or for therapeutic applications and drug delivery.

  12. Characterization of Membrane Integrity and Morphological Stability of Human Salivary Exosomes

    National Research Council Canada - National Science Library

    Kumeda, Nahoko; Ogawa, Yuko; Akimoto, Yoshihiro; Kawakami, Hayato; Tsujimoto, Masafumi; Yanoshita, Ryohei

    2017-01-01

    .... Moreover, intact exosomes could be isolated from whole saliva that had been stored at 4°C. Membrane disruption with detergents such as Triton X-100 and Nonidet P-40 caused partial solubilization of DPP IV and release...

  13. Depletion of microglia and inhibition of exosome synthesis halt tau propagation

    Science.gov (United States)

    Asai, Hirohide; Ikezu, Seiko; Tsunoda, Satoshi; Medalla, Maria; Luebke, Jennifer; Haydar, Tarik; Wolozin, Benjamin; Butovsky, Oleg; Kügler, Sebastian; Ikezu, Tsuneya

    2015-01-01

    Accumulation of pathological tau protein is a major hallmark of Alzheimer’s disease. Tau protein spreads from the entorhinal cortex to the hippocampal region early in the disease. Microglia, the primary phagocytes in the brain, are positively correlated with tau pathology, but their involvement in tau propagation is unknown. We developed an adeno-associated virus–based model exhibiting rapid tau propagation from the entorhinal cortex to the dentate gyrus in 4 weeks. We found that depleting microglia dramatically suppressed the propagation of tau and reduced excitability in the dentate gyrus in this mouse model. Moreover, we demonstrate that microglia spread tau via exosome secretion, and inhibiting exosome synthesis significantly reduced tau propagation in vitro and in vivo. These data suggest that microglia and exosomes contribute to the progression of tauopathy and that the exosome secretion pathway may be a therapeutic target. PMID:26436904

  14. Simplified protocol for flow cytometry analysis of fluorescently labeled exosomes and microvesicles using dedicated flow cytometer

    Czech Academy of Sciences Publication Activity Database

    Pospíchalová, V.; Svoboda, Jan; Dave, Z.; Kotrbová, A.; Kaiser, K.; Klemová, D.; Ilkovics, L.; Hampl, A.; Crha, I.; Jandáková, E.; Minář, L.; Weinberger, V.; Bryja, Vítězslav

    2015-01-01

    Roč. 4, March 31 (2015), s. 25530 ISSN 2001-3078 Institutional support: RVO:61388971 ; RVO:68081707 Keywords : exosomes * microvesicles * extracellular vesicles Subject RIV: EE - Microbiology, Virology

  15. The emerging role of exosomes in Epithelial-Mesenchymal-Transition in cancer.

    Directory of Open Access Journals (Sweden)

    Laura Jayne Vella

    2014-12-01

    Full Text Available Metastasis in cancer consists of multiple steps, including Epithelial-Mesenchymal-Transition (EMT, which is characterized by the loss of Epithelial-like characteristics and the gain of Mesenchymal-like attributes including cell migration and invasion. It is clear that the tumour microenvironment can promote the metastatic cascade and that intercellular communication is necessary for this to occur. Exosomes are small membranous vesicles secreted by most cell types into the extracellular environment and they are important communicators in the tumour microenvironment. They promote angiogenesis, invasion and proliferation in recipient cells to support tumour growth and a prometastatic phenotype. Although it is clear that exosomes contribute to cancer cell plasticity, experimental evidence to define exosome induced plasticity as EMT is only just coming to light. This review will discuss recent research on exosomal regulation of the EMT process in the tumour microenvironment.

  16. Microfluidics for exosome isolation and analysis: enabling liquid biopsy for personalized medicine.

    Science.gov (United States)

    Contreras-Naranjo, Jose C; Wu, Hung-Jen; Ugaz, Victor M

    2017-10-25

    Exosomes, the smallest sized extracellular vesicles (∽30-150 nm) packaged with lipids, proteins, functional messenger RNAs and microRNAs, and double-stranded DNA from their cells of origin, have emerged as key players in intercellular communication. Their presence in bodily fluids, where they protect their cargo from degradation, makes them attractive candidates for clinical application as innovative diagnostic and therapeutic tools. But routine isolation and analysis of high purity exosomes in clinical settings is challenging, with conventional methods facing a number of drawbacks including low yield and/or purity, long processing times, high cost, and difficulties in standardization. Here we review a promising solution, microfluidic-based technologies that have incorporated a host of separation and sensing capabilities for exosome isolation, detection, and analysis, with emphasis on point-of-care and clinical applications. These new capabilities promise to advance fundamental research while paving the way toward routine exosome-based liquid biopsy for personalized medicine.

  17. Exosomes surf on filopodia to enter cells at endocytic hot spots, traffic within endosomes, and are targeted to the ER.

    Science.gov (United States)

    Heusermann, Wolf; Hean, Justin; Trojer, Dominic; Steib, Emmanuelle; von Bueren, Stefan; Graff-Meyer, Alexandra; Genoud, Christel; Martin, Katrin; Pizzato, Nicolas; Voshol, Johannes; Morrissey, David V; Andaloussi, Samir E L; Wood, Matthew J; Meisner-Kober, Nicole C

    2016-04-25

    Exosomes are nanovesicles released by virtually all cells, which act as intercellular messengers by transfer of protein, lipid, and RNA cargo. Their quantitative efficiency, routes of cell uptake, and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake, which saturates with dose and time and reaches near 100% transduction efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. After internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction, and suggest unanticipated routes of subcellular cargo delivery. © 2016 Heusermann et al.

  18. Superhydrophobic surfaces allow probing of exosome self organization using X-ray scattering

    KAUST Repository

    Accardo, Angelo

    2013-01-01

    Drops of exosome dispersions from healthy epithelial colon cell line and colorectal cancer cells were dried on a superhydrophobic PMMA substrate. The residues were studied by small- and wide-angle X-ray scattering using both a synchrotron radiation micrometric beam and a high-flux table-top X-ray source. Structural differences between healthy and cancerous cells were detected in the lamellar lattices of the exosome macro-aggregates. © 2013 The Royal Society of Chemistry.

  19. Effects of embryo-derived exosomes on the development of bovine cloned embryos.

    Science.gov (United States)

    Qu, Pengxiang; Qing, Suzhu; Liu, Ruiqi; Qin, Hongyu; Wang, Weiwei; Qiao, Fang; Ge, Hui; Liu, Jun; Zhang, Yong; Cui, Wei; Wang, Yongsheng

    2017-01-01

    The developmental competence of in vitro cultured (IVC) embryos is markedly lower than that of their in vivo counterparts, suggesting the need for optimization of IVC protocols. Embryo culture medium is routinely replaced three days after initial culture in bovine, however, whether this protocol is superior to continuous nonrenewal culture method under current conditions remains unclear. Using bovine somatic cell nuclear transfer (SCNT) embryos as the model, our results showed that compared with routine renewal treatment, nonrenewal culture system significantly improved blastocyst formation, blastocyst quality (increased total cell number, decreased stress and apoptosis, enhanced Oct-4 expression and ratio of ICM/TE), as well as following development to term. Existence and function of SCNT embryo-derived exosomes were then investigated to reveal the cause of impaired development induced by culture medium replacement. Exosomes were successfully isolated through differential centrifugation and identified by both electron microscopy and immunostaining against exosomal membrane marker CD9. Supplementation of extracted exosomes into freshly renewed medium significantly rescued not only blastocyst formation and quality (in vitro development), but also following growth to term (in vivo development). Notably, ratio of ICM/TE and calving rate were enhanced to a similar level as that in nonrenewal group. In conclusion, our results for the first time indicate that 1: bovine SCNT embryos can secrete exosomes into chemically defined culture medium during IVC; 2: secreted exosomes are essential for SCNT blastocyst formation, blastocyst quality, and following development to term; 3: removal of exosomes induced by culture medium replacement impairs SCNT embryo development, which can be avoided by nonrenewal culture procedure or markedly recovered by exosome supplementation.

  20. The modulation of co-stimulatory molecules by circulating exosomes in primary biliary cirrhosis.

    Science.gov (United States)

    Tomiyama, Takashi; Yang, Guo-Xiang; Zhao, Ming; Zhang, Weici; Tanaka, Hajime; Wang, Jing; Leung, Patrick Sc; Okazaki, Kazuichi; He, Xiao-Song; Lu, Qianjin; Coppel, Ross L; Bowlus, Christopher L; Gershwin, M Eric

    2017-03-01

    Exosomes are nanoparticles of endocytic origin, secreted by a myriad of cell populations that are attracting increased attention by virtue of their ability to modulate cell-to-cell communications. They are also attracting attention in a variety of immunological issues, including autoimmunity and, in particular, their ability to regulate cytokine and chemokine activation. Primary biliary cirrhosis (PBC) is considered a model autoimmune disease, which has a highly focused cytotoxic response against biliary epithelial cells. We have isolated exosomes from plasma from 29 patients with PBC and 30 healthy controls (HCs), and studied the effect of these exosomes on co-stimulatory molecule expression and cytokine production in mononuclear cell populations using an ex vivo system. We also identified the microRNA (miRNA) populations in PBC compared to HC exosomes. We report herein that although exosomes do not change cytokine production, they do significantly alter co-stimulatory molecule expression on antigen-presenting populations. Further, we demonstrated that CD86 up-regulated expression on CD14(+) monocytes, whereas CD40 up-regulated on CD11c(+) dendritic cells by exosomes from patients with PBC. In addition, there were differences of miRNA expression of circulating exosomes in patients with PBC. These data have significant importance based on observations that co-stimulatory molecules play a differential role in the regulation of T-cell activation. Our observation indicated that aberrant exosomes from PBC selectively induce expression of co-stimulatory molecules in different subset of antigen-presenting cells. These alterations may involve in pathogenesis of autoimmune liver disease.Cellular & Molecular Immunology advance online publication, 21 September 2015; doi:10.1038/cmi.2015.86.

  1. Clustering and Candidate Motif Detection in Exosomal miRNAs by Application of Machine Learning Algorithms.

    Science.gov (United States)

    Gaur, Pallavi; Chaturvedi, Anoop

    2017-07-22

    The clustering pattern and motifs give immense information about any biological data. An application of machine learning algorithms for clustering and candidate motif detection in miRNAs derived from exosomes is depicted in this paper. Recent progress in the field of exosome research and more particularly regarding exosomal miRNAs has led much bioinformatic-based research to come into existence. The information on clustering pattern and candidate motifs in miRNAs of exosomal origin would help in analyzing existing, as well as newly discovered miRNAs within exosomes. Along with obtaining clustering pattern and candidate motifs in exosomal miRNAs, this work also elaborates the usefulness of the machine learning algorithms that can be efficiently used and executed on various programming languages/platforms. Data were clustered and sequence candidate motifs were detected successfully. The results were compared and validated with some available web tools such as 'BLASTN' and 'MEME suite'. The machine learning algorithms for aforementioned objectives were applied successfully. This work elaborated utility of machine learning algorithms and language platforms to achieve the tasks of clustering and candidate motif detection in exosomal miRNAs. With the information on mentioned objectives, deeper insight would be gained for analyses of newly discovered miRNAs in exosomes which are considered to be circulating biomarkers. In addition, the execution of machine learning algorithms on various language platforms gives more flexibility to users to try multiple iterations according to their requirements. This approach can be applied to other biological data-mining tasks as well.

  2. RNA substrate length as an indicator of exosome interactions in vivo [version 2; referees: 3 approved

    Directory of Open Access Journals (Sweden)

    Clémentine Delan-Forino

    2017-07-01

    Full Text Available Background: The exosome complex plays key roles in RNA processing and degradation in Eukaryotes and Archaea. Outstanding structural studies identified multiple pathways for RNA substrates into the exosome in vitro, but identifying the pathway followed by individual RNA species in vivo remains challenging. Methods: We attempted to address this question using RNase protection. In vivo RNA-protein crosslinking (CRAC was applied to the exosome component Rrp44/Dis3, which has both endonuclease and exonuclease activity. During CRAC, the exosome was purified under native conditions and subjected to RNase digestion, prior to protein denaturation and cDNA cloning. The resulting high-throughput sequence reads were stratified by length of the cDNA sequence. This should reflect RNA fragment lengths, and therefore the RNA region that was protected by exosome binding. We anticipated major read lengths of ~30nt and ~10nt, reflecting the “central channel” and “direct access” routes to the Rrp44 exonuclease active site observed in vitro. Results: Unexpectedly, no clear peak was observed at 30nt, whereas a broad peak was seen around 20nt. The expected ~10nt peak was seen, and showed strong elevation in strains lacking exonuclease activity. Unexpectedly, this peak was suppressed by point mutations in the Rrp44 endonuclease active site. This indicates that the short fragments are degraded by the exonuclease activity of Rrp44, but also suggests that at least some may be generated by endonuclease activity. Conclusions: The absence of 30nt protected fragments may reflect obligatory binding of cofactors at the entrance to the exosome central channel in vivo. The presence of ~20nt fragments apparently indicates an access route not yet reported from in vitro studies. Confident mapping of 10nt reads is challenging, but they are clearly derived from a subset of exosome targets. In particular, pre-rRNA species, which are major exosome targets, are strongly

  3. Identification and Reconstruction of Prostate Tumor-Suppressing Exosomes for Therapeutic Applications

    Science.gov (United States)

    2016-03-01

    Fevrier B, Raposo G. Exosomes: endosomal-derived vesicles shipping extracellular messages. Current opinion in cell biology 2004;16(4):415-21. 2. Record...overarching challenges of PCRP is to develop effective treatments for advanced prostate cancer. As nano-meter sized vesicles released by many cell types...meter sized vesicles released by many cell types (1). Comprised of lipids, proteins, coding and non-coding RNAs, exosomes serve as cargo carriers

  4. Isolation of Exosomes from Blood Plasma: Qualitative and Quantitative Comparison of Ultracentrifugation and Size Exclusion Chromatography Methods.

    Directory of Open Access Journals (Sweden)

    Tamás Baranyai

    Full Text Available Exosomes are emerging targets for biomedical research. However, suitable methods for the isolation of blood plasma-derived exosomes without impurities have not yet been described.Therefore, we investigated the efficiency and purity of exosomes isolated with potentially suitable methods; differential ultracentrifugation (UC and size exclusion chromatography (SEC.Exosomes were isolated from rat and human blood plasma by various UC and SEC conditions. Efficiency was investigated at serial UC of the supernatant, while in case of SEC by comparing the content of exosomal markers of various fractions. Purity was assessed based on the presence of albumin. We found that the diameter of the majority of isolated particles fell into the size range of exosomes, however, albumin was also present in the preparations, when 1h UC at 4°C was applied. Furthermore, with this method only a minor fraction of total exosomes could be isolated from blood as deduced from the constant amount of exosomal markers CD63 and TSG101 detected after serial UC of rat blood plasma samples. By using UC for longer time or with shorter sedimentation distance at 4°C, or UC performed at 37°C, exosomal yield increased, but albumin impurity was still observed in the isolates, as assessed by transmission electron microscopy, dynamic light scattering and immunoblotting against CD63, TSG101 and albumin. Efficiency and purity were not different in case of using further diluted samples. By using SEC with different columns, we have found that although a minor fraction of exosomes can be isolated without significant albumin content on Sepharose CL-4B or Sephacryl S-400 columns, but not on Sepharose 2B columns, the majority of exosomes co-eluted with albumin.Here we show that it is feasible to isolate exosomes from blood plasma by SEC without significant albumin contamination albeit with low vesicle yield.

  5. Exosomes Derived from Dendritic Cells Treated with Schistosoma japonicum Soluble Egg Antigen Attenuate DSS-Induced Colitis

    Directory of Open Access Journals (Sweden)

    Lifu Wang

    2017-09-01

    Full Text Available Exosomes are 30–150 nm small membrane vesicles that are released into the extracellular medium via cells that function as a mode of intercellular communication. Dendritic cell (DC-derived exosomes modulate immune responses and prevent the development of autoimmune diseases. Moreover, Schistosoma japonicum eggs show modulatory effects in a mouse model of colitis. Therefore, we hypothesized that exosomes derived from DCs treated with S. japonicum soluble eggs antigen (SEA; SEA-treated DC exosomes would be useful for treating inflammatory bowel disease (IBD. Exosomes were purified from the supernatant of DCs treated or untreated with SEA and identified via transmission electron microscopy, western blotting and NanoSight. Acute colitis was induced via the administration of dextran sulfate sodium (DSS in drinking water (5.0%, wt/vol. Treatment with exosomes was conducted via intraperitoneal injection (i.p.; 50 μg per mouse from day 0 to day 6. Clinical scores were calculated based on weight loss, stool type, and bleeding. Colon length was measured as an indirect marker of inflammation, and colon macroscopic characteristics were determined. Body weight loss and the disease activity index of DSS-induced colitis mice decreased significantly following treatment with SEA-treated DC exosomes. Moreover, the colon lengths of SEA-treated DC exosomes treated colitis mice improved, and their mean colon macroscopic scores decreased. In addition, histologic examinations and histological scores showed that SEA-treated DC exosomes prevented colon damage in acute DSS-induced colitis mice. These results indicate that SEA-treated DC exosomes attenuate the severity of acute DSS-induced colitis mice more effectively than DC exosomes. The current work suggests that SEA-treated DC exosomes may be useful as a new approach to treat IBD.

  6. Cancer becomes wasteful: emerging roles of exosomes in cell-fate determination

    Directory of Open Access Journals (Sweden)

    Franz Wendler

    2013-09-01

    Full Text Available Extracellular vesicles (EVs, including exosomes, have been widely recognized for their role in intercellular communication of the immune response system. In the past few years, significance has been given to exosomes in the induction and modulation of cell-fate-inducing signalling pathways, such as the Hedgehog (Hh, Wnts, Notch, transforming growth factor (TGF-β, epidermal growth factor (EGF and fibroblast growth factor (FGF pathways, placing them in the wider context of development and also of cancer. These protein families induce signalling cascades responsible for tissue specification, homeostasis and maintenance. Exosomes contribute to cell-fate signal secretion, and vice versa exosome secretion can be induced by these proteins. Interestingly, exosomes can also transfer their mRNA to host cells or modulate the signalling pathways directly by the removal of downstream effector molecules from the cell. Surprisingly, much of what we know about the function of exosomes in cell determination is gathered from pathological transformed cancer cells and wound healing while data about their biogenesis and biology in normal developing and adult tissue lag behind. In this report, we will summarize some of the published literature and point to current advances and questions in this fast-developing topic. In a brief foray, we will also update and shortly discuss their potential in diagnosis and targeted cancer treatment.

  7. ATF1 and RAS in exosomes are potential clinical diagnostic markers for cervical cancer.

    Science.gov (United States)

    Shi, Yanhua; Wang, Wei; Yang, Baozhi; Tian, Hongge

    2017-10-01

    Cervical cancer is one of the most common cancers among women worldwide. It is highly lethal yet can be treated when found in early stage. Thus, early detection is of significant important for early diagnosis of cervical cancer. Exosomes have been used as biomarkers in clinical diagnosis. It is unknown that whether blood exosomes associated with cervical cancer can be detected and if these exosomes can accurately represent the developmental stage of cervical cancer. Mouse models were made out of a relapsed cervical cancer patient's tumour sample for original and recurrent cervical cancer, and gene analysis in both tumours and exosomes in these mouse models were performed. We found that activating transcription factor 1 (ATF1) and RAS genes were significantly up-regulated in tumours of both primary and recurrent cervical cancer mouse model, and they can also be detected in the blood exosomes of the mouse model. Our results indicated that ATF1 and RAS could be potential candidate biomarkers for cervical cancer in early diagnosis. ATF1 and RAS genes were found significantly elevated in tumours of primary and recurrent cervical cancer mouse model, and they were also detected in the blood exosomes. Therefore, ATF1 and RAS could be used as a diagnostic marker for cervical cancer in the future. Copyright © 2017 John Wiley & Sons, Ltd.

  8. Lactation-Related MicroRNA Expression Profiles of Porcine Breast Milk Exosomes

    Science.gov (United States)

    Liang, Yan; Zhong, Zhijun; Wang, Xiaoyan; Zhou, Qi; Chen, Lei; Lang, Qiulei; He, Zhiping; Chen, Xiaohui; Gong, Jianjun; Gao, Xiaolian; Li, Xuewei; Lv, Xuebin

    2012-01-01

    Breast milk is the primary source of nutrition for newborns, and is rich in immunological components. MicroRNAs (miRNAs) are present in various body fluids and are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and could regulate target gene expression and recipient cell function. Here, we analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth) by a deep sequencing. We found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10−16, χ2 test) and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk-only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are a prelude to in-depth investigations of the essential roles of breast milk in the development of the infant’s immune system. PMID:22937080

  9. Characterization of the Proinflammatory Profile of Synovial Fluid-Derived Exosomes of Patients with Osteoarthritis

    Directory of Open Access Journals (Sweden)

    Rossana Domenis

    2017-01-01

    Full Text Available The purpose of this study is to characterize synovial fluid- (SF- derived exosomes of patients with gonarthrosis comparing two methods of isolation and to investigate their immune regulatory properties. Extracellular vesicles (EVs have been isolated from inflamed SF by polymer precipitation method and quantified by Exocet kit and by nanoparticle tracking analysis. Vesicles expressed all the specific exosomal markers by immunoblot and FACS. After isolation with Exoquick, a relevant contamination by immune complexes was detected, which required further magnetic bead-based purification to remove. SF-derived exosomes significantly stimulated the release of several inflammatory cytokines and chemokines and metalloproteinases by M1 macrophages but did not influence the expression of CD80 and CD86 costimulatory molecules. In conclusion, we characterized purified exosomes isolated from inflamed SF and demonstrate that purified exosomes are functionally active in their ability to stimulate the release of proinflammatory factors from M1 macrophages. Our data indicate that SF-derived exosomes from gonarthrosis patients play a role in disease progression.

  10. Lactation-related microRNA expression profiles of porcine breast milk exosomes.

    Directory of Open Access Journals (Sweden)

    Yiren Gu

    Full Text Available Breast milk is the primary source of nutrition for newborns, and is rich in immunological components. MicroRNAs (miRNAs are present in various body fluids and are selectively packaged inside the exosomes, a type of membrane vesicles, secreted by most cell types. These exosomal miRNAs could be actively delivered into recipient cells, and could regulate target gene expression and recipient cell function. Here, we analyzed the lactation-related miRNA expression profiles in porcine milk exosomes across the entire lactation period (newborn to 28 days after birth by a deep sequencing. We found that immune-related miRNAs are present and enriched in breast milk exosomes (p<10(-16, χ(2 test and are generally resistant to relatively harsh conditions. Notably, these exosomal miRNAs are present in higher numbers in the colostrums than in mature milk. It was higher in the serum of colostrum-only fed piglets compared with the mature milk-only fed piglets. These immune-related miRNA-loaded exosomes in breast milk may be transferred into the infant body via the digestive tract. These observations are a prelude to in-depth investigations of the essential roles of breast milk in the development of the infant's immune system.

  11. Raman spectroscopy and SERS analysis of ovarian tumour derived exosomes (TEXs): a preliminary study

    Science.gov (United States)

    Kerr, Laura T.; Gubbins, Luke; Weiner Gorzel, Karolina; Sharma, Shiva; Kell, Malcolm; McCann, Amanda; Hennelly, Bryan M.

    2014-05-01

    Here we report a preliminary study based on the application of Raman spectroscopy and surface enhanced Raman spectroscopy (SERS) to investigate the compositional differences between exosomes derived from ovarian carcinoma cells (cell line A2780) grown in normoxia (normal O2 conditions) and hypoxia (1% O2 conditions). Exosomes are integral to cell signalling, and are of interest in the study of how cells communicate within their environment. We are particularly interested in identifying whether hypoxia induced senescent cells can communi- cate via exosomes with neighbouring tumour cells, thereby causing them to become senescent and therefore radio and chemo resistant. With this goal in mind, we performed a preliminary study on the application of Raman spectroscopy and SERS to analyse the biomolecular fingerprint of both groups of exosomes and to investigate whether there exists a different biomolecular composition associated with exosomes derived from hypoxic cells in comparison to those from normoxic cells. We also applied multivariate statistical techniques for the classification of both groups of exosomes.

  12. Exosomes as novel bio-carriers for gene and drug delivery.

    Science.gov (United States)

    Jiang, Xin-Chi; Gao, Jian-Qing

    2017-04-15

    Clinical treatments have stalled in certain diseases due to a lack of proper therapeutic delivery systems. Recent studies have identified exosomes for their potential use as cell-free therapies, which may provide a novel mechanism for solving this problem. Exosomes are nanoscale extracellular vesicles that can transport rich cargos of proteins, lipids, DNA, and RNA. It is increasingly recognized that exosomes play a complex role in not only the physiological conditions but also pathological ones. Accumulating evidence suggests that exosomes are of paramount importance in distant cell-cell communication because they can enter the circulation when secreted and pass through additional biological barriers. As a result, interest has exploded surrounding the functional parameters of exosomes and their potential applications as delivery vehicles for small molecule therapies. In this review, we discuss the potential of exosomes to be utilized as "natural nanoparticles" to deliver drugs and genes, and their advantages and disadvantages are compared to other delivery mechanisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Global scientific trends on exosome research during 2007-2016: a bibliometric analysis.

    Science.gov (United States)

    Wang, Yiran; Wang, Qijin; Wei, Xianzhao; Shao, Jie; Zhao, Jian; Zhang, Zicheng; Chen, Ziqiang; Bai, Yushu; Wang, Ning; Wang, Yajie; Li, Ming; Zhai, Xiao

    2017-07-18

    Exosomes are small vesicles of endosomal origin, and they can be used for the diagnosis and the treatment. However, limited data were for the evaluation of the trend of exosome researches. This study aims to investigate the trend of exosome researches and compare the contribution of research from different regions, organizations and authors. Exosome related publications from 2007 to 2016 were retrieved from the Web of Science database. Excel, GraphPad Prism 5 and VOSviewer software were used to analyze the research trend. A total of 1852 papers were identified and were cited 62967 times. The United States accounted for 38.8% of the articles, 42.0% of the citations, and the highest H-index (76). China ranked the second in the number of articles, but the sixth in citation frequency (4337) and the fourth in H-index (36). The journals, PLoS ONE and J Biol Chem had the highest number of publications. The author, Gabrielsson S., has published the most papers in this field (22). The keyword "ribonucleic acid" was mentioned the most at 746 times, and the words, "stem cell", "drug resistance" and "monocyte cell factor" were the latest hotspots appeared around 2015. Literature growth related to exosome is expanding rapidly. The quality of the articles from China still requires improvement. Recent studies focus on the relationship with tumor, and "stem cell", "drug resistance" and "michigan cancer foundation-7" may be the newest topics that should be closely followed in exosome research.

  14. Differential expression of exosomal microRNAs in prefrontal cortices of schizophrenia and bipolar disorder patients.

    Directory of Open Access Journals (Sweden)

    Meredith G Banigan

    Full Text Available Exosomes are cellular secretory vesicles containing microRNAs (miRNAs. Once secreted, exosomes are able to attach to recipient cells and release miRNAs potentially modulating the function of the recipient cell. We hypothesized that exosomal miRNA expression in brains of patients diagnosed with schizophrenia (SZ and bipolar disorder (BD might differ from controls, reflecting either disease-specific or common aberrations in SZ and BD patients. The sources of the analyzed samples included McLean 66 Cohort Collection (Harvard Brain Tissue Resource Center, BrainNet Europe II (BNE, a consortium of 18 brain banks across Europe and Boston Medical Center (BMC. Exosomal miRNAs from frozen postmortem prefrontal cortices with well-preserved RNA were isolated and submitted to profiling by Luminex FLEXMAP 3D microfluidic device. Multiple statistical analyses of microarray data suggested that certain exosomal miRNAs were differentially expressed in SZ and BD subjects in comparison to controls. RT-PCR validation confirmed that two miRNAs, miR-497 in SZ samples and miR-29c in BD samples, have significantly increased expression when compared to control samples. These results warrant future studies to evaluate the potential of exosome-derived miRNAs to serve as biomarkers of SZ and BD.

  15. Exosomes As Potential Biomarkers and Targeted Therapy in Colorectal Cancer: A Mini-Review

    Directory of Open Access Journals (Sweden)

    Kha Wai Hon

    2017-08-01

    Full Text Available The number of colorectal cancer (CRC cases have increased gradually year by year. In fact, CRC is one of the most widely diagnosed cancer in men and women today. This disease is usually diagnosed at a later stage of the development, and by then, the chance of survival has declined significantly. Even though substantial progress has been made in understanding the basic molecular mechanism of CRC, there is still a lack of understanding in using the available information for diagnosing CRC effectively. Liquid biopsies are minimally invasive and have become the epitome of a good screening source for stage-specific diagnosis, measuring drug response and severity of the disease. There are various circulating entities that can be found in biological fluids, and among them, exosomes, have been gaining considerable attention. Exosomes can be found in almost all biological fluids including serum, urine, saliva, and breast milk. Furthermore, exosomes carry valuable molecular information such as proteins and nucleic acids that directly reflects the source of the cells. Nevertheless, the inconsistent yield and isolation process and the difficulty in obtaining pure exosomes have become major obstacles that need to be addressed. The potential usage of exosomes as biomarkers have not been fully validated and explored yet. This review attempts to uncover the potential molecules that can be derived from CRC-exosomes as promising biomarkers or molecular targets for effective diagnosing of CRC.

  16. Structure of an Rrp6-RNA exosome complex bound to poly(A) RNA

    Energy Technology Data Exchange (ETDEWEB)

    Wasmuth, Elizabeth V.; Januszyk, Kurt; Lima, Christopher D. [MSKCC

    2014-08-20

    The eukaryotic RNA exosome processes and degrades RNA by directing substrates to the distributive or processive 3' to 5' exoribonuclease activities of Rrp6 or Rrp44, respectively. The non-catalytic nine-subunit exosome core (Exo9) features a prominent central channel. Although RNA can pass through the channel to engage Rrp44, it is not clear how RNA is directed to Rrp6 or whether Rrp6 uses the central channel. Here we report a 3.3 Å crystal structure of a ten-subunit RNA exosome complex from Saccharomyces cerevisiae composed of the Exo9 core and Rrp6 bound to single-stranded poly(A) RNA. The Rrp6 catalytic domain rests on top of the Exo9 S1/KH ring above the central channel, the RNA 3' end is anchored in the Rrp6 active site, and the remaining RNA traverses the S1/KH ring in an opposite orientation to that observed in a structure of a Rrp44-containing exosome complex. Solution studies with human and yeast RNA exosome complexes suggest that the RNA path to Rrp6 is conserved and dependent on the integrity of the S1/KH ring. Although path selection to Rrp6 or Rrp44 is stochastic in vitro, the fate of a particular RNA may be determined in vivo by the manner in which cofactors present RNA to the RNA exosome.

  17. Role of TIM-4 in exosome-dependent entry of HIV-1 into human immune cells.

    Science.gov (United States)

    Sims, Brian; Farrow, Anitra L; Williams, Sparkle D; Bansal, Anju; Krendelchtchikov, Alexandre; Gu, Linlin; Matthews, Qiana L

    2017-01-01

    Exosomes, 30-200 nm nanostructures secreted from donor cells and internalized by recipient cells, can play an important role in the cellular entry of some viruses. These microvesicles are actively secreted into various body fluids, including blood, urine, saliva, cerebrospinal fluid, and breast milk. We successfully isolated exosomes from human breast milk and plasma. The size and concentration of purified exosomes were measured by nanoparticle tracking, while Western blotting confirmed the presence of the exosomal-associated proteins CD9 and CD63, clathrin, and T cell immunoglobulin and mucin proteins (TIMs). Through viral infection assays, we determined that HIV-1 utilizes an exosome-dependent mechanism for entry into human immune cells. The virus contains high amounts of phosphatidylserine (PtdSer) and may bind PtdSer receptors, such as TIMs. This mechanism is supported by our findings that exosomes from multiple sources increased HIV-1 entry into T cells and macrophages, and viral entry was potently blocked with anti-TIM-4 antibodies.

  18. Quantifying exosome secretion from single cells reveals a modulatory role for GPCR signaling.

    Science.gov (United States)

    Verweij, Frederik Johannes; Bebelman, Maarten P; Jimenez, Connie R; Garcia-Vallejo, Juan J; Janssen, Hans; Neefjes, Jacques; Knol, Jaco C; de Goeij-de Haas, Richard; Piersma, Sander R; Baglio, S Rubina; Verhage, Matthijs; Middeldorp, Jaap M; Zomer, Anoek; van Rheenen, Jacco; Coppolino, Marc G; Hurbain, Ilse; Raposo, Graça; Smit, Martine J; Toonen, Ruud F G; van Niel, Guillaume; Pegtel, D Michiel

    2018-01-16

    Exosomes are small endosome-derived extracellular vesicles implicated in cell-cell communication and are secreted by living cells when multivesicular bodies (MVBs) fuse with the plasma membrane (PM). Current techniques to study exosome physiology are based on isolation procedures after secretion, precluding direct and dynamic insight into the mechanics of exosome biogenesis and the regulation of their release. In this study, we propose real-time visualization of MVB-PM fusion to overcome these limitations. We designed tetraspanin-based pH-sensitive optical reporters that detect MVB-PM fusion using live total internal reflection fluorescence and dynamic correlative light-electron microscopy. Quantitative analysis demonstrates that MVB-PM fusion frequency is reduced by depleting the target membrane SNAREs SNAP23 and syntaxin-4 but also can be induced in single cells by stimulation of the histamine H1 receptor (H1HR). Interestingly, activation of H1R1 in HeLa cells increases Ser110 phosphorylation of SNAP23, promoting MVB-PM fusion and the release of CD63-enriched exosomes. Using this single-cell resolution approach, we highlight the modulatory dynamics of MVB exocytosis that will help to increase our understanding of exosome physiology and identify druggable targets in exosome-associated pathologies. © 2018 Verweij et al.

  19. Exosomes derived from gastric cancer cells activate NF-κB pathway in macrophages to promote cancer progression.

    Science.gov (United States)

    Wu, Lijun; Zhang, Xu; Zhang, Bin; Shi, Hui; Yuan, Xiao; Sun, Yaoxiang; Pan, Zhaoji; Qian, Hui; Xu, Wenrong

    2016-09-01

    Exosomes are nano-sized membrane vesicles secreted by both normal and cancer cells. Emerging evidence indicates that cancer cells derived exosomes contribute to cancer progression through the modulation of tumor microenvironment. However, the effects of exosomes derived from gastric cancer cells on macrophages are not well understood. In this study, we investigated the biological role of gastric cancer cells derived exosomes in the activation of macrophages. We demonstrated that gastric cancer cells derived exosomes activated macrophages to express increased levels of proinflammatory factors, which in turn promoted tumor cell proliferation and migration. In addition, gastric cancer cells derived exosomes remarkably upregulated the phosphorylation of NF-κB in macrophages. Inhibiting the activation of NF-κB reversed the upregulation of proinflammatory factors in macrophages and blocked their promoting effects on gastric cancer cells. Moreover, we found that gastric cancer cells derived exosomes could also activate macrophages from human peripheral blood monocytes through the activation of NF-κB. In conclusion, our results suggest that gastric cancer cells derived exosomes stimulate the activation of NF-κB pathway in macrophages to promote cancer progression, which provides a potential therapeutic approach for gastric cancer by interfering with the interaction between exosomes and macrophages in tumor microenvironment.

  20. Phosphoproteome Characterization of Human Colorectal Cancer SW620 Cell-Derived Exosomes and New Phosphosite Discovery for C-HPP.

    Science.gov (United States)

    Guo, Jiahui; Cui, Yizhi; Yan, Ziqi; Luo, Yanzhang; Zhang, Wanling; Deng, Suyuan; Tang, Shengquan; Zhang, Gong; He, Qing-Yu; Wang, Tong

    2016-11-04

    Identification of all phosphorylation forms of known proteins is a major goal of the Chromosome-Centric Human Proteome Project (C-HPP). Recent studies have found that certain phosphoproteins can be encapsulated in exosomes and function as key regulators in tumor microenvironment, but no deep coverage phosphoproteome of human exosomes has been reported to date, which makes the exosome a potential source for the new phosphosite discovery. In this study, we performed highly optimized MS analyses on the exosomal and cellular proteins isolated from human colorectal cancer SW620 cells. With stringent data quality control, 313 phosphoproteins with 1091 phosphosites were confidently identified from the SW620 exosome, from which 202 new phosphosites were detected. Exosomal phosphoproteins were significantly enriched in the 11q12.1-13.5 region of chromosome 11 and had a remarkably high level of tyrosine-phosphorylated proteins (6.4%), which were functionally relevant to ephrin signaling pathway-directed cytoskeleton remodeling. In conclusion, we here report the first high-coverage phosphoproteome of human cell-secreted exosomes, which leads to the identification of new phosphosites for C-HPP. Our findings provide insights into the exosomal phosphoprotein systems that help to understand the signaling language being delivered by exosomes in cell-cell communications. The mass spectrometry proteomics data have been deposited to the ProteomeXchange consortium with the data set identifier PXD004079, and iProX database (accession number: IPX00076800).

  1. Kinome profiling of non-canonical TRAIL signaling reveals RIP1-Src-STAT3-dependent invasion in resistant non-small cell lung cancer cells

    NARCIS (Netherlands)

    Azijli, Kaamar; Yuvaraj, Saravanan; Peppelenbosch, Maikel P.; Wurdinger, Thomas; Dekker, Henk; Joore, Jos; van Dijk, Evert; Quax, Wim J.; Peters, Godefridus J.; de Jong, Steven; Kruyt, Frank A. E.

    2012-01-01

    Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) triggers apoptosis selectively in tumor cells through interaction with TRAIL-R1/DR4 or TRAIL-R2/DR5 and this process is considered a promising avenue for cancer treatment. TRAIL resistance, however, is frequently encountered and

  2. Human IL6 stimulates bovine satellite cell proliferation through a Signal transducer and activator of transcription 3 (STAT3)-dependent mechanism.

    Science.gov (United States)

    Brandt, A M; Kania, J M; Reinholt, B M; Johnson, S E

    2018-01-01

    Bovine satellite cell (bSC) myogenesis and skeletal muscle hypertrophy occur through the orchestrated actions of multiple autocrine and paracrine growth factors. Intimate to the bSC niche is IL6, a dual-purpose cytokine with proinflammatory and mitogenic properties. The objective of the experiment was to examine the effects of IL6 on proliferation and differentiation of bSC in vitro. Treatment of primary bSC cultures with recombinant bovine IL6 (bIL6) failed to alter myogenesis owing to the absence of intracellular signal transduction. The cytokine was able to stimulate phosphorylation of signal transducer and activator of transcription 3 tyrosine 705 (STAT3Y705) in Madin-Darby bovine kidney (MDBK) epithelial cells, thus demonstrating bioactivity. Media supplemented with recombinant human IL6 (hIL6) caused phosphorylation of STAT3Y705 in bSC and increased (P bSC proliferation. Morphologic and biochemical measures of bSC differentiation remained unchanged (P > 0.05) following treatment for 48 h with hIL6. These results support a role for hIL6 as a bSC mitogen in vitro. The inability of bIL6 to initiate an intracellular signal in bSC requires further investigation. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. IL-10 induces a STAT3-dependent autoregulatory loop in TH2 cells that promotes Blimp-1 restriction of cell expansion via antagonism of STAT5 target genes.

    Science.gov (United States)

    Poholek, Amanda C; Jankovic, Dragana; Villarino, Alejandro V; Petermann, Franziska; Hettinga, Angela; Shouval, Dror S; Snapper, Scott B; Kaech, Susan M; Brooks, Stephen R; Vahedi, Golnaz; Sher, Alan; Kanno, Yuka; O'Shea, John J

    2016-10-01

    Blimp-1 expression in T cells extinguishes the fate of T follicular helper cells, drives terminal differentiation, and limits autoimmunity. Although various factors have been described to control Blimp-1 expression in T cells, little is known about what regulates Blimp-1 expression in T helper 2 (TH2) cells and the molecular basis of its actions. We report that signal transducer and activator of transcription 3 (STAT3) unexpectedly played a critical role in regulating Blimp-1 in TH2 cells. Furthermore, we found that the cytokine interleukin-10 (IL-10) acted directly on TH2 cells and was necessary and sufficient to induce optimal Blimp-1 expression through STAT3. Together, Blimp-1 and STAT3 amplified IL-10 production in TH2 cells, creating a strong autoregulatory loop that enhanced Blimp-1 expression. Increased Blimp-1 in T cells antagonized STAT5-regulated cell cycle and antiapoptotic genes to limit cell expansion. These data elucidate the signals required for Blimp-1 expression in TH2 cells and reveal an unexpected mechanism of action of IL-10 in T cells, providing insights into the molecular underpinning by which Blimp-1 constrains T cell expansion to limit autoimmunity.

  4. Exosomes and other extracellular vesicles in host–pathogen interactions

    Science.gov (United States)

    Schorey, Jeffrey S; Cheng, Yong; Singh, Prachi P; Smith, Victoria L

    2015-01-01

    An effective immune response requires the engagement of host receptors by pathogen-derived molecules and the stimulation of an appropriate cellular response. Therefore, a crucial factor in our ability to control an infection is the accessibility of our immune cells to the foreign material. Exosomes—which are extracellular vesicles that function in intercellular communication—may play a key role in the dissemination of pathogen- as well as host-derived molecules during infection. In this review, we highlight the composition and function of exosomes and other extracellular vesicles produced during viral, parasitic, fungal and bacterial infections and describe how these vesicles could function to either promote or inhibit host immunity. PMID:25488940

  5. Exosomes in the nose induce immune cell trafficking and harbour an altered protein cargo in chronic airway inflammation.

    Science.gov (United States)

    Lässer, Cecilia; O'Neil, Serena E; Shelke, Ganesh V; Sihlbom, Carina; Hansson, Sara F; Gho, Yong Song; Lundbäck, Bo; Lötvall, Jan

    2016-06-20

    Exosomes are nano-sized extracellular vesicles participating in cell-to-cell communication both in health and disease. However, the knowledge about the functions and molecular composition of exosomes in the upper airways is limited. The aim of the current study was therefore to determine whether nasal exosomes can influence inflammatory cells and to establish the proteome of nasal lavage fluid-derived exosomes in healthy subjects, as well as its alterations in individuals with chronic airway inflammatory diseases [asthma and chronic rhinosinusitis (CRS)]. Nasal lavage fluid was collected from 14 healthy subjects, 15 subjects with asthma and 13 subjects with asthma/CRS. Exosomes were isolated with differential centrifugation and the proteome was analysed by LC-MS/MS with the application of two exclusion lists as well as using quantitative proteomics. Ingenuity Pathways Analysis and GO Term finder was used to predict the functions associated with the exosomal proteome and a migration assay was used to analyse the effect on immune cells by nasal exosomes. Firstly, we demonstrate that nasal exosomes can induce migration of several immune cells, such as monocytes, neutrophils and NK cells in vitro. Secondly, a mass spectrometry approach, with the application of exclusion lists, was utilised to generate a comprehensive protein inventory of the exosomes from healthy subjects. The use of exclusion lists resulted in the identification of ~15 % additional proteins, and increased the confidence in ~20 % of identified proteins. In total, 604 proteins were identified in nasal exosomes and the nasal exosomal proteome showed strong associations with immune-related functions, such as immune cell trafficking. Thirdly, a quantitative proteomics approach was used to determine alterations in the exosome proteome as a result of airway inflammatory disease. Serum-associated proteins and mucins were more abundant in the exosomes from subjects with respiratory diseases compared to

  6. Investigation of Content, Stoichiometry and Transfer of miRNA from Human Neural Stem Cell Line Derived Exosomes.

    Directory of Open Access Journals (Sweden)

    Lara Stevanato

    Full Text Available Exosomes are small (30-100 nm membrane vesicles secreted by a variety of cell types and only recently have emerged as a new avenue for cell-to-cell communication. They are natural shuttles of RNA and protein cargo, making them attractive as potential therapeutic delivery vehicles. MicroRNAs (miRNAs are short non-coding RNAs which regulate biological processes and can be found in exosomes. Here we characterized the miRNA contents of exosomes derived from human neural stem cells (hNSCs. Our investigated hNSC line is a clonal, conditionally immortalized cell line, compliant with good manufacturing practice (GMP, and in clinical trials for stroke and critical limb ischemia in the UK (clinicaltrials.gov: NCT01151124, NCT02117635, and NCT01916369. By using next generation sequencing (NGS technology we identified the presence of a variety of miRNAs in both exosomal and cellular preparations. Many of these miRNAs were enriched in exosomes indicating that cells specifically sort them for extracellular release. Although exosomes have been proven to contain miRNAs, the copy number quantification per exosome of a given miRNA remains unclear. Herein we quantified by real-time PCR a highly shuttled exosomal miRNA subtype (hsa-miR-1246 in order to assess its stoichiometry per exosome. Furthermore, we utilized an in vitro system to confirm its functional transfer by measuring the reduction in luciferase expression using a 3' untranslated region dual luciferase reporter assay. In summary, NGS analysis allowed the identification of a unique set of hNSC derived exosomal miRNAs. Stoichiometry and functional transfer analysis of one of the most abundant identified miRNA, hsa-miR-1246, were measured to support biological relevance of exosomal miRNA delivery.

  7. Exosomal lipids impact notch signaling and induce death of human pancreatic tumoral SOJ-6 cells.

    Directory of Open Access Journals (Sweden)

    Sadia Beloribi

    Full Text Available Exosomes are of increasing interest as alternative mode of cell-to-cell communication. We previously reported that exosomes secreted by human SOJ-6 pancreatic tumor cells induce (glycoprotein ligand-independent cell death and inhibit Notch-1 pathway, this latter being particularly active during carcinogenesis and in cancer stem cells. Therefore, we asked whether exosomal lipids were key-elements for cell death and hypothesized that cholesterol-rich membrane microdomains were privileged sites of exosome interactions with tumor cells. To address these questions and based on the lipid composition of exosomes from SOJ-6 cells (Ristorcelli et al. (2008 FASEB J. 22; 3358-3369 enriched in cholesterol and sphingomyelin (lipids forming liquid-ordered phase, Lo and depleted in phospholipids (lipids forming liquid-disordered phase, Ld, we designed Synthetic Exosome-Like Nanoparticles (SELN with ratios Lo/Ld from 3.0 to 6.0 framing that of SOJ-6 cell exosomes. SELN decreased tumor cell survival, the higher the Lo/Ld ratio, the lower the cell survival. This decreased survival was due to activation of cell death with inhibition of Notch pathway. FRET analyses indicated fusions/exchanges of SELN with cell membranes. Fluorescent SELN co-localized with the ganglioside GM1 then with Rab5A, markers of lipid microdomains and of early endosomes, respectively. These interactions occurred at lipid microdomains of plasma and/or endosome membranes where the Notch-1 pathway matures. We thus demonstrated a major role for lipids in interactions between SELN and tumor cells, and in the ensued cell death. To our knowledge this is the first report on such effects of lipidic nanoparticles on tumor cell behavior. This may have implications in tumor progression.

  8. Exosomes from Human Dental Pulp Stem Cells Suppress Carrageenan-Induced Acute Inflammation in Mice.

    Science.gov (United States)

    Pivoraitė, Ugnė; Jarmalavičiūtė, Akvilė; Tunaitis, Virginijus; Ramanauskaitė, Giedrė; Vaitkuvienė, Aida; Kašėta, Vytautas; Biziulevičienė, Genė; Venalis, Algirdas; Pivoriūnas, Augustas

    2015-10-01

    The primary goal of this study was to examine the effects of human dental pulp stem cell-derived exosomes on the carrageenan-induced acute inflammation in mice. Exosomes were purified by differential ultracentrifugation from the supernatants of stem cells derived from the dental pulp of human exfoliated deciduous teeth (SHEDs) cultivated in serum-free medium. At 1 h post-carrageenan injection, exosomes derived from supernatants of 2 × 10(6) SHEDs were administered by intraplantar injection to BALB/c mice; 30 mg/kg of prednisolone and phosphate-buffered saline (PBS) were used as positive and negative controls, respectively. Edema was measured at 6, 24, and 48 h after carrageenan injection. For the in vivo imaging experiments, AngioSPARK750, Cat B 750 FAST, and MMPSense 750 FAST were administered into the mouse tail vein 2 h post-carrageenan injection. Fluorescence images were acquired at 6, 24, and 48 h after edema induction by IVIS Spectrum in vivo imaging system. Exosomes significantly reduced the carrageenan-induced edema at all the time points studied (by 39.5, 41.6, and 25.6% at 6, 24, and 48 h after injection, respectively), to similar levels seen with the positive control (prednisolone). In vivo imaging experiments revealed that, both exosomes and prednisolone suppress activities of cathepsin B and matrix metalloproteinases (MMPs) at the site of carrageenan-induced acute inflammation, showing more prominent effects of prednisolone at the early stages, while exosomes exerted their suppressive effects gradually and at later time points. Our study demonstrates for the first time that exosomes derived from human dental pulp stem cells suppress carrageenan-induced acute inflammation in mice.

  9. Concise Review: MSC-Derived Exosomes for Cell-Free Therapy.

    Science.gov (United States)

    Phinney, Donald G; Pittenger, Mark F

    2017-04-01

    Mesenchymal stem cell transplantation is undergoing extensive evaluation as a cellular therapy in human clinical trials. Because MSCs are easily isolated and amenable to culture expansion in vitro there is a natural desire to test MSCs in many diverse clinical indications. This is exemplified by the rapidly expanding literature base that includes many in vivo animal models. More recently, MSC-derived extracellular vesicles (EVs), which include exosomes and microvesicles (MV), are being examined for their role in MSC-based cellular therapy. These vesicles are involved in cell-to-cell communication, cell signaling, and altering cell or tissue metabolism at short or long distances in the body. The exosomes and MVs can influence tissue responses to injury, infection, and disease. MSC-derived exosomes have a content that includes cytokines and growth factors, signaling lipids, mRNAs, and regulatory miRNAs. To the extent that MSC exosomes can be used for cell-free regenerative medicine, much will depend on the quality, reproducibility, and potency of their production, in the same manner that these parameters dictate the development of cell-based MSC therapies. However, the MSC exosome's contents are not static, but rather a product of the MSC tissue origin, its activities and the immediate intercellular neighbors of the MSCs. As such, the exosome content produced by MSCs appears to be altered when MSCs are cultured with tumor cells or in the in vivo tumor microenvironment. Therefore, careful attention to detail in producing MSC exosomes may provide a new therapeutic paradigm for cell-free MSC-based therapies with decreased risk. Stem Cells 2017;35:851-858. © 2017 AlphaMed Press.

  10. Exosomes: vehicles for the transfer of toxic proteins associated with neurodegenerative diseases?

    Directory of Open Access Journals (Sweden)

    Shayne Anthony Bellingham

    2012-05-01

    Full Text Available Exosomes are small membranous vesicles secreted by a number of cell types including neurons and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of endosomes to form multivesicular bodies (MVB. When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell-cell signaling, removal of unwanted proteins, and the transfer of pathogens between cells. One such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD of humans or bovine spongiform encephalopathy (BSE of cattle. Similarly, exosomes are also involved in the processing of the amyloid precursor protein (APP which is associated with Alzheimer's disease (AD. Exosomes have been shown to contain full-length APP and several distinct proteolytically cleaved products of APP, including Aβ. In addition, these fragments can be modulated using inhibitors of the proteases involved in APP cleavage. These observations provide further evidence for a novel pathway in which PrP and APP fragments are released from cells. Other proteins such as superoxide dismutase I (SOD-1 and alpha-synuclein (involved in Amyotrophic Lateral Sclerosis (ALS and Parkinson’s disease respectively are also found associated with exosomes. This review will focus on the role of exosomes in neurodegenerative disorders and discuss the potential of these vesicles for the spread of neurotoxicity, therapeutics and diagnostics for these diseases.

  11. Altered lysosomal proteins in neural-derived plasma exosomes in preclinical Alzheimer disease.

    Science.gov (United States)

    Goetzl, Edward J; Boxer, Adam; Schwartz, Janice B; Abner, Erin L; Petersen, Ronald C; Miller, Bruce L; Kapogiannis, Dimitrios

    2015-07-07

    Diverse autolysosomal proteins were quantified in neurally derived blood exosomes from patients with Alzheimer disease (AD) and controls to investigate disordered neuronal autophagy. Blood exosomes obtained once from patients with AD (n = 26) or frontotemporal dementia (n = 16), other patients with AD (n = 20) both when cognitively normal and 1 to 10 years later when diagnosed, and case controls were enriched for neural sources by anti-human L1CAM antibody immunoabsorption. Extracted exosomal proteins were quantified by ELISAs and normalized with the CD81 exosomal marker. Mean exosomal levels of cathepsin D, lysosome-associated membrane protein 1 (LAMP-1), and ubiquitinylated proteins were significantly higher and of heat-shock protein 70 significantly lower for AD than controls in cross-sectional studies (p ≤ 0.0005). Levels of cathepsin D, LAMP-1, and ubiquitinylated protein also were significantly higher for patients with AD than for patients with frontotemporal dementia (p ≤ 0.006). Step-wise discriminant modeling of the protein levels correctly classified 100% of patients with AD. Exosomal levels of all proteins were similarly significantly different from those of matched controls in 20 patients 1 to 10 years before and at diagnosis of AD (p ≤ 0.0003). Levels of autolysosomal proteins in neurally derived blood exosomes distinguish patients with AD from case controls and appear to reflect the pathology of AD up to 10 years before clinical onset. These preliminary results confirm in living patients with AD the early appearance of neuronal lysosomal dysfunction and suggest that these proteins may be useful biomarkers in large prospective studies. © 2015 American Academy of Neurology.

  12. Feedback activation of STAT3 mediates trastuzumab resistance via upregulation of MUC1 and MUC4 expression

    Science.gov (United States)

    Li, Wei; Fan, Kexing; Qian, Weizhu; Hou, Sheng; Wang, Hao; Dai, Jianxin; Wei, Huafeng; Guo, Yajun

    2014-01-01

    Although HER2-targeting antibody trastuzumab confers a substantial benefit for patients with HER2-overexpressing breast and gastric cancer, overcoming trastuzumab resistance remains a large unmet need. In this study, we revealed a STAT3-centered positive feedback loop that mediates the resistance of trastuzumab. Mechanistically, chronic exposure of trastuzumab causes the upregulation of fibronection (FN), EGF and IL-6 in parental trastuzumab-sensitive breast and gastric cells and convergently leads to STAT3 hyperactivation. Activated STAT3 enhances the expression of FN, EGF and IL-6, thus constituting a positive feedback loop which amplifies and maintains the STAT3 signal; furthermore, hyperactivated STAT3 signal promotes the expression of MUC1 and MUC4, consequently mediating trastuzumab resistance via maintenance of persistent HER2 activation and masking of trastuzumab binding to HER2 respectively. Genetic or pharmacological inhibition of STAT3 disrupted STAT3-dependent positive feedback loop and recovered the trastuzumab sensitivity partially due to increased apoptosis induction. Combined trastuzumab with STAT3 inhibition synergistically suppressed the growth of the trastuzumab-resistant tumor xenografts in vivo. Taken together, our results suggest that feedback activation of STAT3 constitutes a key node mediating trastuzumab resistance. Combinatorial targeting on both HER2 and STAT3 may enhance the efficacy of trastuzumab or other HER2-targeting agents in HER2-positive breast and gastric cancer. PMID:25327561

  13. Air2p is critical for the assembly and RNA-binding of the TRAMP complex and the KOW domain of Mtr4p is crucial for exosome activation

    Science.gov (United States)

    Holub, Peter; Lalakova, Jana; Cerna, Hana; Pasulka, Josef; Sarazova, Marie; Hrazdilova, Kristyna; Arce, Maria Sanudo; Hobor, Fruzsina; Stefl, Richard; Vanacova, Stepanka

    2012-01-01

    Trf4/5p-Air1/2p-Mtr4p polyadenylation complex (TRAMP) is an essential component of nuclear RNA surveillance in yeast. It recognizes a variety of nuclear transcripts produced by all three RNA polymerases, adds short poly(A) tails to aberrant or unstable RNAs and activates the exosome for their degradation. Despite the advances in understanding the structural features of the isolated complex subunits or their fragments, the details of complex assembly, RNA recognition and exosome activation remain poorly understood. Here we provide the first understanding of the RNA binding mode of the complex. We show that Air2p is an RNA-binding subunit of TRAMP. We identify the zinc knuckles (ZnK) 2, 3 and 4 as the RNA-binding domains, and reveal the essentiality of ZnK4 for TRAMP4 polyadenylation activity. Furthermore, we identify Air2p as the key component of TRAMP4 assembly providing bridging between Mtr4p and Trf4p. The former is bound via the N-terminus of Air2p, while the latter is bound via ZnK5, the linker between ZnK4 and 5 and the C-terminus of the protein. Finally, we uncover the RNA binding part of the Mtr4p arch, the KOW domain, as the essential component for TRAMP-mediated exosome activation. PMID:22402490

  14. Multifaceted effects of oligodendroglial exosomes on neurons: impact on neuronal firing rate, signal transduction and gene regulation.

    Science.gov (United States)

    Fröhlich, Dominik; Kuo, Wen Ping; Frühbeis, Carsten; Sun, Jyh-Jang; Zehendner, Christoph M; Luhmann, Heiko J; Pinto, Sheena; Toedling, Joern; Trotter, Jacqueline; Krämer-Albers, Eva-Maria

    2014-09-26

    Exosomes are small membranous vesicles of endocytic origin that are released by almost every cell type. They exert versatile functions in intercellular communication important for many physiological and pathological processes. Recently, exosomes attracted interest with regard to their role in cell-cell communication in the nervous system. We have shown that exosomes released from oligodendrocytes upon stimulation with the neurotransmitter glutamate are internalized by neurons and enhance the neuronal stress tolerance. Here, we demonstrate that oligodendroglial exosomes also promote neuronal survival during oxygen-glucose deprivation, a model of cerebral ischaemia. We show the transfer from oligodendrocytes to neurons of superoxide dismutase and catalase, enzymes which are known to help cells to resist oxidative stress. Additionally, we identify various effects of oligodendroglial exosomes on neuronal physiology. Electrophysiological analysis using in vitro multi-electrode arrays revealed an increased firing rate of neurons exposed to oligodendroglial exosomes. Moreover, gene expression analysis and phosphorylation arrays uncovered differentially expressed genes and altered signal transduction pathways in neurons after exosome treatment. Our study thus provides new insight into the broad spectrum of action of oligodendroglial exosomes and their effects on neuronal physiology. The exchange of extracellular vesicles between neural cells may exhibit remarkable potential to impact brain performance. © 2014 The Author(s) Published by the Royal Society. All rights reserved.

  15. Structure of the nuclear exosome component Rrp6p reveals an interplay between the active site and the HRDC domain

    DEFF Research Database (Denmark)

    Midtgaard, Søren Fuglsang; Assenholt, Jannie; Jonstrup, Anette Thyssen

    2006-01-01

    The multisubunit eukaryotic exosome is an essential RNA processing and degradation machine. In its nuclear form, the exosome associates with the auxiliary factor Rrp6p, which participates in both RNA processing and degradation reactions. The crystal structure of Saccharomyces cerevisiae Rrp6p...

  16. The Role of Isolation Methods on a Nanoscale Surface Structure and its Effect on the Size of Exosomes