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1
Maire, Cecile, Amanda Salviano-Silva, Katharina Kolbe, Manfred Westphal, Katrin Lamszus, and Franz Ricklefs. "TMIC-64. EXTRACELLULAR VESICLE TRAFFICKING IN GBM." Neuro-Oncology 24, Supplement_7 (November 1, 2022): vii285—vii286. http://dx.doi.org/10.1093/neuonc/noac209.1108.
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Abstract Extracellular vesicles (EVs) are secreted by all cell types, including tumor cells, and are found in increased numbers in the plasma of GBM patients. EVs may contain high-value genetic material that can be useful for tracking tumor development, as well as membrane proteins that affect other cells. This prompted us to investigate how tumor EVs might influence immune cells in glioma, and in primary and secondary lymphoid organs as well as in the circulation. To this end we used a syngeneic GBM mouse model and tracked tumor EVs from the brain to the meninges, cervical lymph nodes, plasma, bone marrow and spleen. Interestingly, we were able to identify tumor EVs mostly in the cervical lymph nodes by ImageStream imaging flow cytometry just 30min after tumor EV injection into the brain. However, when tumor EVs were produced by a large gliomas transfected with dTomato, we found them mainly in plasma, less frequently in bone marrow and never in the spleen. We confirmed these data by extracting DNA from EVs and detecting specific dTomato sequences using digital droplet PCR. In addition, we detected CD11b+ macrophages in the meninges that likely travel through the lymphatics that have taken up tumor EV or tumor material. We confirm that tumor EVs are capable of eliciting an immune response by activating T cells. However, prolonged contact and large number of EVs could also block antigen recognition by T cells and thus contribute to the propagation of an immunosuppressive environment in GBM.
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Kumar, Prashant, Fahad Zadjali, Ying Yao, Michael Köttgen, Alexis Hofherr, Kenneth W. Gross, Darshan Mehta, and John J. Bissler. "Single Gene Mutations in Pkd1 or Tsc2 Alter Extracellular Vesicle Production and Trafficking." Biology 11, no. 5 (May 6, 2022): 709. http://dx.doi.org/10.3390/biology11050709.
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Patients with autosomal dominant polycystic kidney disease (ADPKD) and tuberous sclerosis complex (TSC) are born with normal or near-normal kidneys that later develop cysts and prematurely lose function. Both renal cystic diseases appear to be mediated, at least in part, by disease-promoting extracellular vesicles (EVs) that induce genetically intact cells to participate in the renal disease process. We used centrifugation and size exclusion chromatography to isolate the EVs for study. We characterized the EVs using tunable resistive pulse sensing, dynamic light scattering, transmission electron microscopy, and Western blot analysis. We performed EV trafficking studies using a dye approach in both tissue culture and in vivo studies. We have previously reported that loss of the Tsc2 gene significantly increased EV production and here demonstrate that the loss of the Pkd1 gene also significantly increases EV production. Using a cell culture system, we also show that loss of either the Tsc2 or Pkd1 gene results in EVs that exhibit an enhanced uptake by renal epithelial cells and a prolonged half-life. Loss of the primary cilia significantly reduces EV production in renal collecting duct cells. Cells that have a disrupted Pkd1 gene produce EVs that have altered kinetics and a prolonged half-life, possibly impacting the duration of the EV cargo effect on the recipient cell. These results demonstrate the interplay between primary cilia and EVs and support a role for EVs in polycystic kidney disease pathogenesis.
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Santamaria, Sara, Maria Cristina Gagliani, Grazia Bellese, Silvia Marconi, Anastasia Lechiara, Martina Dameri, Cinzia Aiello, Erica Tagliatti, Patrizio Castagnola, and Katia Cortese. "Imaging of Endocytic Trafficking and Extracellular Vesicles Released Under Neratinib Treatment in ERBB2+ Breast Cancer Cells." Journal of Histochemistry & Cytochemistry 69, no. 7 (June 15, 2021): 461–73. http://dx.doi.org/10.1369/00221554211026297.
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Breast cancers (BCa) with ERBB2 amplification show rapid tumor growth, increased disease progression, and lower survival rate. Deregulated intracellular trafficking and extracellular vesicle (EVs) release are mechanisms that support cancer progression and resistance to treatments. Neratinib (NE) is a Food and Drug Administration–approved pan-ERBB inhibitor employed for the treatment of ERBB2+ BCa that blocks signaling and causes survival inhibition. However, the effects of NE on ERBB2 internalization, its trafficking to multivesicular bodies (MVBs), and the release of EVs that originate from these organelles remain poorly studied. By confocal and electron microscopy, we observed that low nanomolar doses of NE induced a modest ERBB2 internalization along with an increase of clathrin-mediated endocytosis and of the CD63+ MVB compartment in SKBR-3 cells. Furthermore, we showed in the culture supernatant two distinct EV subsets, based on their size and ERBB2 positivity: small (30–100 nm) ERBB2− EVs and large (>100 nm) ERBB2+ EVs. In particular, we found that NE increased the overall release of EVs, which displayed a reduced ERBB2 positivity compared with controls. Taken together, these results provide novel insight into the effects of NE on ERBB2+ BCa cells that may lead to a reduction of ERBB2 potentially transferred to distant target cells by EVs:
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Baxter, Amy A. "Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking." International Journal of Molecular Sciences 21, no. 19 (October 1, 2020): 7256. http://dx.doi.org/10.3390/ijms21197256.
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Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.
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Roberts-Dalton, H. D., A. Cocks, J. M. Falcon-Perez, E. J. Sayers, J. P. Webber, P. Watson, A. Clayton, and A. T. Jones. "Fluorescence labelling of extracellular vesicles using a novel thiol-based strategy for quantitative analysis of cellular delivery and intracellular traffic." Nanoscale 9, no. 36 (2017): 13693–706. http://dx.doi.org/10.1039/c7nr04128d.
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Ortega, Miguel A., Oscar Fraile-Martinez, Cielo Garcia-Montero, Miguel Angel Alvarez-Mon, Ana Maria Gomez-Lahoz, Agustin Albillos, Guillermo Lahera, et al. "An Updated View of the Importance of Vesicular Trafficking and Transport and Their Role in Immune-Mediated Diseases: Potential Therapeutic Interventions." Membranes 12, no. 6 (May 25, 2022): 552. http://dx.doi.org/10.3390/membranes12060552.
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Cellular trafficking is the set of processes of distributing different macromolecules by the cell. This process is highly regulated in cells, involving a system of organelles (endomembranous system), among which are a great variety of vesicles that can be secreted from the cell, giving rise to different types of extracellular vesicles (EVs) that can be captured by other cells to modulate their function. The cells of the immune system are especially sensitive to this cellular traffic, producing and releasing different classes of EVs, especially in disease states. There is growing interest in this field due to the therapeutic and translational possibilities it offers. Different ways of taking advantage of the understanding of cell trafficking and EVs are being investigated, and their use as biomarkers or therapeutic targets is being investigated. The objective of this review is to collect the latest results and knowledge in this area with a specific focus on immune-mediated diseases. Although some promising results have been obtained, further knowledge is still needed, at both the basic and translational levels, to understand and modulate cellular traffic and EVs for better clinical management of these patients.
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Cruz Camacho, Abel, Daniel Alfandari, Ewa Kozela, and Neta Regev-Rudzki. "Biogenesis of extracellular vesicles in protozoan parasites: The ESCRT complex in the trafficking fast lane?" PLOS Pathogens 19, no. 2 (February 23, 2023): e1011140. http://dx.doi.org/10.1371/journal.ppat.1011140.
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Extracellular vesicles (EVs) provide a central mechanism of cell–cell communication. While EVs are found in most organisms, their pathogenesis-promoting roles in parasites are of particular interest given the potential for medical insight and consequential therapeutic intervention. Yet, a key feature of EVs in human parasitic protozoa remains elusive: their mechanisms of biogenesis. Here, we survey the current knowledge on the biogenesis pathways of EVs secreted by the four main clades of human parasitic protozoa: apicomplexans, trypanosomatids, flagellates, and amoebae. In particular, we shine a light on findings pertaining to the Endosomal Sorting Complex Required for Transport (ESCRT) machinery, as in mammals it plays important roles in EV biogenesis. This review highlights the diversity in EV biogenesis in protozoa, as well as the related involvement of the ESCRT system in these unique organisms.
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Zhang, Pan, Su Bin Lim, Kuan Jiang, Ti Weng Chew, Boon Chuan Low, and Chwee Teck Lim. "Distinct mRNAs in Cancer Extracellular Vesicles Activate Angiogenesis and Alter Transcriptome of Vascular Endothelial Cells." Cancers 13, no. 9 (April 22, 2021): 2009. http://dx.doi.org/10.3390/cancers13092009.
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Cancer-derived extracellular vesicles (EVs) have been demonstrated to be implicated in various processes of cancer development, with most of the EV-induced changes attributed to EV-proteins and EV-microRNAs. However, the knowledge about the abundance of cancer EV-mRNAs and their contribution to cancer development remain elusive. Here, we show that mRNAs prevail in cancer EVs as compared with normal EVs, and cancer EVs that carry abundant angiogenic mRNAs activate angiogenesis in human umbilical vein endothelial cells (HUVECs). Specifically, of a gene panel comprising 61 hypoxia-targeted oncogenes, a larger proportion is harbored by cancer EVs (>40%) than normal EVs (14.8%). Fluorescent trafficking indicates cancer EVs deliver translatable mRNAs such as VEGFA to HUVECs, contributing to the activation of VEGFR-dependent angiogenesis and the upregulation of epithelial-mesenchymal transition-related and metabolism-related genes. Overall, our findings provide novel insights into EV-mRNAs and their role in angiogenesis, and has potential for diagnostic and therapeutic applications.
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Auger, Clément, Aude Brunel, Tiffany Darbas, Hussein Akil, Aurélie Perraud, Gaëlle Bégaud, Barbara Bessette, Niki Christou, and Mireille Verdier. "Extracellular Vesicle Measurements with Nanoparticle Tracking Analysis: A Different Appreciation of Up and Down Secretion." International Journal of Molecular Sciences 23, no. 4 (February 19, 2022): 2310. http://dx.doi.org/10.3390/ijms23042310.
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As is the case with most eucaryotic cells, cancer cells are able to secrete extracellular vesicles (EVs) as a communication means towards their environment and surrounding cells. EVs are represented by microvesicles and smaller vesicles called exosomes, which are known for their involvement in cancer aggressiveness. The release of such EVs requires the intervention of trafficking-associated proteins, mostly represented by the RAB-GTPases family. In particular, RAB27A is known for its role in addressing EVs-to-be secreted towards the the plasma membrane. In this study, shRNAs targeting RAB27A were used in colorectal (CRC) and glioblastoma (GB) cell lines in order to alter EVs secretion. To study and monitor EVs secretion in cell lines’ supernatants, nanoparticle tracking analysis (NTA) was used through the NanoSight NS300 device. Since it appeared that NanoSight failed to detect the decrease in the EVs secretion, we performed another approach to drop EVs secretion (RAB27A-siRNA, indomethacin, Nexihnib20). Similar results were obtained i.e., no variation in EVs concentration. Conversely, NTA allowed us to monitor EVs up-secretion following rotenone treatment or hypoxia conditions. Therefore, our data seemed to point out the insufficiency of using only this technique for the assessment of EVs secretion decrease.
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Lipinski, Simone, and Katharina Tiemann. "Extracellular Vesicles and Their Role in the Spatial and Temporal Expansion of Tumor–Immune Interactions." International Journal of Molecular Sciences 22, no. 7 (March 25, 2021): 3374. http://dx.doi.org/10.3390/ijms22073374.
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Extracellular vesicles (EVs) serve as trafficking vehicles and intercellular communication tools. Their cargo molecules directly reflect characteristics of their parental cell. This includes information on cell identity and specific cellular conditions, ranging from normal to pathological states. In cancer, the content of EVs derived from tumor cells is altered and can induce oncogenic reprogramming of target cells. As a result, tumor-derived EVs compromise antitumor immunity and promote cancer progression and spreading. However, this pro-oncogenic phenotype is constantly being challenged by EVs derived from the local tumor microenvironment and from remote sources. Here, we summarize the role of EVs in the tumor–immune cross-talk that includes, but is not limited to, immune cells in the tumor microenvironment. We discuss the potential of remotely released EVs from the microbiome and during physical activity to shape the tumor–immune cross-talk, directly or indirectly, and confer antitumor activity. We further discuss the role of proinflammatory EVs in the temporal development of the tumor–immune interactions and their potential use for cancer diagnostics.
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Picca, Anna, Flora Guerra, Riccardo Calvani, Cecilia Bucci, Maria Lo Monaco, Anna Bentivoglio, Hélio Coelho-Júnior, Francesco Landi, Roberto Bernabei, and Emanuele Marzetti. "Mitochondrial Dysfunction and Aging: Insights from the Analysis of Extracellular Vesicles." International Journal of Molecular Sciences 20, no. 4 (February 13, 2019): 805. http://dx.doi.org/10.3390/ijms20040805.
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The progressive decline of cell function and integrity, manifesting clinically as increased vulnerability to adverse outcomes and death, is core to biological aging. Mitochondrial dysfunction, oxidative stress, altered intercellular communication (including chronic low-grade inflammation), genomic instability, telomere attrition, loss of proteostasis, altered nutrient sensing, epigenetic alterations, and stem cell exhaustion have been proposed as hallmarks of aging. These “aging pillars” are not mutually exclusive, making the matter intricate and leaving numerous unanswered questions. The characterization of circulating extracellular vesicles (EVs) has recently allowed specific secretory phenotypes associated with aging to be identified. As such, EVs may serve as novel biomarkers for capturing the complexity of aging. Besides the mitochondrial–lysosomal axis, EV trafficking has been proposed as an additional layer in mitochondrial quality control. Indeed, disruption of the mitochondrial–lysosomal axis coupled with abnormal EV secretion may play a role in the pathogenesis of aging and several disease conditions. Here, we discuss (1) the mechanisms of EV generation; (2) the relationship between the mitochondrial–lysosomal axis and EV trafficking in the setting of mitochondrial quality control; and (3) the prospect of using EVs as aging biomarkers and as delivery systems for therapeutics against age-related conditions.
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Abdelhamed, Sherif, Noah I. Hornick, and Peter Kurre. "Residual HSPC in the Leukemia Microenvironment Are Reprogrammed Via Extracellular Vesicle Trafficking." Blood 128, no. 22 (December 2, 2016): 888. http://dx.doi.org/10.1182/blood.v128.22.888.888.
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Several groups have shown that leukemic cells create a self-reinforcing bone marrow (BM) niche that functionally impairs normal hematopoietic stem and progenitor cells (HSPC) indirectly through stroma-secreted factors. We recently demonstrated an alternative mechanism whereby extracellular vesicles (EVs) from acute myeloid leukemia (AML) patients and cell lines, but not BM CD34 controls, suppress their clonogenicity through EV trafficking of microRNA that directly downregulate critical transcription factors (c-Myb and HoxA9). Here, we aimed to clarify the fate of residual HSPC in in vivo AML xenografts, as well as ex vivo intrafemural (IF) injection and in vitro exposure of EVs experiments. Among KSL cells we observed a significant increase in the frequency of the long-term hematopoietic stem cell (SLAM, CD150+CD48−) subpopulation, but not the multipotent progenitors even at low levels of AML infiltration or direct IF injection of EVs. The HSPC pool redistribution was accompanied by cell cycle alterations in residual HSPC that showed AML EVs consistently induced quiescence (G0) in KSL (cKit+Sca1+Lin−) HSPC populations. When we assessed their DNA damage, residual HSPC showed a distinct increase in the gH2AX foci relative to control non-engrafted mice as well as the transcriptional upregulation of Rad51 and P21 genes along with gains in phosphorylation of the tumor suppressor p53. Yet, the reprogrammed KSL showed no evidence of apoptosis indicated by the lack of upregulation of the p53 target, Puma, and Annexin V staining, nor evidence of senescence (P16 and Sparc transcripts). To gain additional insight, we performed a tandem mass tag (TMT) proteomic profiling of AML-EV exposed HSPC with or without exposure to EVs derived from AML cells. The results showed significant enrichment of DNA methylation regulatory pathway such as DNMT1, HELLS and UHRF1 as well as inflammatory pathways including IL1b, NOS, CEBPB and NFkB pathway-targets, confirmed by transcriptional profiling of KSL from xeno-transplanted mice. Based on our recent report that miR-1246 is one of the most highly enriched miRNA in AML derived EVs and proceeded to determine its target transcripts using an attenuated RISC complex (RISC-Trap), followed by high-throughput sequencing. Bioinformatics analysis identified a set of 27 miR-1246-specific targets relative to control microRNAs. Strikingly, the target set was selectively enriched for a panel of negative cell-cycle regulator genes (CDK1, CDK7, CDK11, CCNF, HDAC2 and GATA3) as well as the DNA methylation regulators (DNMT1 and HELLS).Collectively, our results demonstrated that residual HSPC in the AML BM are phenotypically reprogrammed and suppressed in their proliferation along with DNA damage accumulation via paracrine EV microRNA trafficking. Our study provides insight into HSPC fates in the AML niche and echoes observations of cell competition, as a mode of non-cell autonomous regulation where p53 activation in the reprogrammed cells leads to a progressive decline in proliferation and fitness. We propose that AML EV trafficking of miR-1246 specifically may contribute to the altered fate of residual HSPC via transcriptional regulation of proliferation-related genes. Disclosures No relevant conflicts of interest to declare.
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Yamamoto, Satoshi, Kohji Okamura, Risa Fujii, Takamasa Kawano, Koji Ueda, Yasutomo Yajima, and Kiyotaka Shiba. "Specimen-specific drift of densities defines distinct subclasses of extracellular vesicles from human whole saliva." PLOS ONE 16, no. 4 (April 8, 2021): e0249526. http://dx.doi.org/10.1371/journal.pone.0249526.
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Extracellular vesicles (EVs) in body fluids constitute heterogenous populations, which mirror their diverse parental cells as well as distinct EV-generation pathways. Various methodologies have been proposed to differentiate EVs in order to deepen the current understanding of EV biology. Equilibrium density-gradient centrifugation has often been used to separate EVs based on their buoyant densities; however, the standard conditions used for the method do not necessarily allow all EVs to move to their equilibrium density positions, which complicates the categorization of EVs. Here, by prolonging ultracentrifugation time to 96 h and fractionating EVs both by floating up or spinning down directions, we allowed 111 EV-associated protein markers from the whole saliva of three healthy volunteers to attain equilibrium. Interestingly, the determined buoyant densities of the markers drifted in a specimen-specific manner, and drift patterns differentiated EVs into at least two subclasses. One class carried classical exosomal markers, such as CD63 and CD81, and the other was characterized by the molecules involved in membrane remodeling or vesicle trafficking. Distinct patterns of density drift may represent the differences in generation pathways of EVs.
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Sawaged, Savannah, Thomas Mota, Honit Piplani, Reetu Thakur, Deepti Lall, Elizabeth McCabe, Soojung Seo, et al. "TBK1 and GABARAP family members suppress Coxsackievirus B infection by limiting viral production and promoting autophagic degradation of viral extracellular vesicles." PLOS Pathogens 18, no. 8 (August 31, 2022): e1010350. http://dx.doi.org/10.1371/journal.ppat.1010350.
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Host-pathogen dynamics are constantly at play during enteroviral infection. Coxsackievirus B (CVB) is a common juvenile enterovirus that infects multiple organs and drives inflammatory diseases including acute pancreatitis and myocarditis. Much like other enteroviruses, CVB is capable of manipulating host machinery to hijack and subvert autophagy for its benefit. We have previously reported that CVB triggers the release of infectious extracellular vesicles (EVs) which originate from autophagosomes. These EVs facilitate efficient dissemination of infectious virus. Here, we report that TBK1 (Tank-binding kinase 1) suppresses release of CVB-induced EVs. TBK1 is a multimeric kinase that directly activates autophagy adaptors for efficient cargo recruitment and induces type-1 interferons during viral-mediated STING recruitment. Positioning itself at the nexus of pathogen elimination, we hypothesized that loss of TBK1 could exacerbate CVB infection due to its specific role in autophagosome trafficking. Here we report that infection with CVB during genetic TBK1 knockdown significantly increases viral load and potentiates the bulk release of viral EVs. Similarly, suppressing TBK1 with small interfering RNA (siRNA) caused a marked increase in intracellular virus and EV release, while treatment in vivo with the TBK1-inhibitor Amlexanox exacerbated viral pancreatitis and EV spread. We further demonstrated that viral EV release is mediated by the autophagy modifier proteins GABARAPL1 and GABARAPL2 which facilitate autophagic flux. We observe that CVB infection stimulates autophagy and increases the release of GABARAPL1/2-positive EVs. We conclude that TBK1 plays additional antiviral roles by inducing autophagic flux during CVB infection independent of interferon signaling, and the loss of TBK1 better allows CVB-laden autophagosomes to circumvent lysosomal degradation, increasing the release of virus-laden EVs. This discovery sheds new light on the mechanisms involved in viral spread and EV propagation during acute enteroviral infection and highlights novel intracellular trafficking protein targets for antiviral therapy.
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Kwok, Zhi Hao, Chenghao Wang, and Yang Jin. "Extracellular Vesicle Transportation and Uptake by Recipient Cells: A Critical Process to Regulate Human Diseases." Processes 9, no. 2 (January 31, 2021): 273. http://dx.doi.org/10.3390/pr9020273.
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Emerging evidence highlights the relevance of extracellular vesicles (EVs) in modulating human diseases including but not limited to cancer, inflammation, and neurological disorders. EVs can be found in almost all types of human body fluids, suggesting that their trafficking may allow for their targeting to remote recipient cells. While molecular processes underlying EV biogenesis and secretion are increasingly elucidated, mechanisms governing EV transportation, target finding and binding, as well as uptake into recipient cells remain to be characterized. Understanding the specificity of EV transport and uptake is critical to facilitating the development of EVs as valuable diagnostics and therapeutics. In this mini review, we focus on EV uptake mechanisms and specificities, as well as their implications in human diseases.
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Di Rocco, Giuliana, Silvia Baldari, and Gabriele Toietta. "Towards Therapeutic Delivery of Extracellular Vesicles: Strategies forIn VivoTracking and Biodistribution Analysis." Stem Cells International 2016 (2016): 1–12. http://dx.doi.org/10.1155/2016/5029619.
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Extracellular vesicles (EVs), such as microvesicles and exosomes, are membranous structures containing bioactive material released by several cells types, including mesenchymal stem/stromal cells (MSCs). Increasing lines of evidences point to EVs as paracrine mediators of the beneficial effects on tissue remodeling associated with cell therapy. Administration of MSCs-derived EVs has therefore the potential to open new and safer therapeutic avenues, alternative to cell-based approaches, for degenerative diseases. However, an enhanced knowledge aboutin vivoEVs trafficking upon delivery is required before effective clinical translation. Only a few studies have focused on the biodistribution analysis of exogenously administered MSCs-derived EVs. Nevertheless, current strategies forin vivotracking in animal models have provided valuable insights on the biodistribution upon systemic delivery of EVs isolated from several cellular sources, indicating in liver, spleen, and lungs the preferential target organs. Different strategies for targeting EVs to specific tissues to enhance their therapeutic efficacy and reduce possible off-target effects have been investigated. Here, in the context of a possible clinical application of MSC-derived EVs for tissue regeneration, we review the existing strategies forin vivotracking and targeting of EVs isolated from different cellular sources and the studies elucidating the biodistribution of exogenously administered EVs.
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Bitencourt, Tamires A., André M. Pessoni, Bianca T. M. Oliveira, Lysangela R. Alves, and Fausto Almeida. "The RNA Content of Fungal Extracellular Vesicles: At the “Cutting-Edge” of Pathophysiology Regulation." Cells 11, no. 14 (July 13, 2022): 2184. http://dx.doi.org/10.3390/cells11142184.
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The role of extracellular vesicles (EVs) in interkingdom communication is widely accepted, and their role in intraspecies communication has been strengthened by recent research. Based on the regulation promoted by EV-associated molecules, the interactions between host and pathogens can reveal different pathways that ultimately affect infection outcomes. As a great part of the regulation is ascribable to RNA contained in EVs, many studies have focused on profiling RNAs in fungal and host EVs, tracking their accumulation during infection, and identifying potential target genes. Herein, we overview the main classes of RNA contained in fungal EVs and the biological processes regulated by these molecules, portraying a state-of-the-art picture of RNAs loaded in fungal EVs, while also raising several questions to drive future investigations. Our compiled data show unambiguously that EVs act as key elements in signaling pathways, and play a crucial role in pathosystems. A complete understanding of the processes that govern RNA content loading and trafficking, and its effect on recipient cells, will lead to improved technologies to ward off infectious agents that threaten human health.
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Soares, Maria, Maria M. Pinto, Rui Jorge Nobre, Luís Pereira de Almeida, Maria da Graça Rasteiro, Teresa Almeida-Santos, João Ramalho-Santos, and Ana Paula Sousa. "Isolation of Extracellular Vesicles from Human Follicular Fluid: Size-Exclusion Chromatography versus Ultracentrifugation." Biomolecules 13, no. 2 (February 2, 2023): 278. http://dx.doi.org/10.3390/biom13020278.
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Follicular fluid (FF) is the microenvironment where a growing oocyte develops. Intrafollicular communication ensures oocyte competence and is carried out through paracrine signaling, the exchange of molecules via gap junctions, and the trafficking of extracellular vesicles (EVs). The study of FF-derived EVs is important for both translational and fundamental research in the female reproductive field. This study aimed to compare the efficacy and purity of two EV isolation methods: size-exclusion chromatography (SEC) and ultracentrifugation (UC). EVs isolated using SEC and UC were compared regarding their size and concentration using dynamic light scattering (DLS) and nanoparticle tracking analysis (NTA); protein contamination was assessed with microBCA; specific EV markers were detected with Western blot, and EV morphology was studied with transmission electron microscopy (TEM). Our results show that although both techniques isolated small EVs, a significantly increased yield in particle number was clear with UC compared with SEC. On the other hand, SEC generated purer EVs with fewer protein contaminants and aggregates. In conclusion, the selection of the most suited approach to isolate EVs must be conducted considering the degree of recovery, purity, and downstream application of the isolated EVs.
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Tse, Shun Wilford, Chee Fan Tan, Jung Eun Park, JebaMercy Gnanasekaran, Nikhil Gupta, Jee Keem Low, Kheng Wei Yeoh, et al. "Microenvironmental Hypoxia Induces Dynamic Changes in Lung Cancer Synthesis and Secretion of Extracellular Vesicles." Cancers 12, no. 10 (October 11, 2020): 2917. http://dx.doi.org/10.3390/cancers12102917.
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Extracellular vesicles (EVs) mediate critical intercellular communication within healthy tissues, but are also exploited by tumour cells to promote angiogenesis, metastasis, and host immunosuppression under hypoxic stress. We hypothesize that hypoxic tumours synthesize hypoxia-sensitive proteins for packing into EVs to modulate their microenvironment for cancer progression. In the current report, we employed a heavy isotope pulse/trace quantitative proteomic approach to study hypoxia sensitive proteins in tumour-derived EVs protein. The results revealed that hypoxia stimulated cells to synthesize EVs proteins involved in enhancing tumour cell proliferation (NRSN2, WISP2, SPRX1, LCK), metastasis (GOLM1, STC1, MGAT5B), stemness (STC1, TMEM59), angiogenesis (ANGPTL4), and suppressing host immunity (CD70). In addition, functional clustering analyses revealed that tumour hypoxia was strongly associated with rapid synthesis and EV loading of lysosome-related hydrolases and membrane-trafficking proteins to enhance EVs secretion. Moreover, lung cancer-derived EVs were also enriched in signalling molecules capable of inducing epithelial-mesenchymal transition in recipient cancer cells to promote their migration and invasion. Together, these data indicate that lung-cancer-derived EVs can act as paracrine/autocrine mediators of tumorigenesis and metastasis in hypoxic microenvironments. Tumour EVs may, therefore, offer novel opportunities for useful biomarkers discovery and therapeutic targeting of different cancer types and at different stages according to microenvironmental conditions.
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Auger, Clément, Niki Christou, Aude Brunel, Aurélie Perraud, and Mireille Verdier. "Autophagy and Extracellular Vesicles in Colorectal Cancer: Interactions and Common Actors?" Cancers 13, no. 5 (March 2, 2021): 1039. http://dx.doi.org/10.3390/cancers13051039.
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Autophagy is a homeostatic process involved in the degradation of disabled proteins and organelles using lysosomes. This mechanism requires the recruitment of specialized proteins for vesicle trafficking, that may also be involved in other types of machinery such as the biogenesis and secretion of extracellular vesicles (EVs), and particularly small EVs called exosomes. Among these proteins, Rab-GTPases may operate in both pathways, thus representing an interesting avenue for further study regarding the interaction between autophagy and extracellular vesicle machinery. Both mechanisms are involved in the development of colorectal cancer (CRC), particularly in cancer stem cell (CSC) survival and communication, although they are not specific to CRC or CSCs. This highlights the importance of studying the crosstalk between autophagy and EVs biogenesis and release.
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Willysson, Annie, Anne-lie Ståhl, Daniel Gillet, Julien Barbier, Jean-Christophe Cintrat, Valérie Chambon, Anne Billet, Ludger Johannes, and Diana Karpman. "Shiga Toxin Uptake and Sequestration in Extracellular Vesicles Is Mediated by Its B-Subunit." Toxins 12, no. 7 (July 10, 2020): 449. http://dx.doi.org/10.3390/toxins12070449.
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Shiga toxin (Stx)-stimulated blood cells shed extracellular vesicles (EVs) which can transfer the toxin to the kidneys and lead to hemolytic uremic syndrome. The toxin can be taken up by renal cells within EVs wherein the toxin is released, ultimately leading to cell death. The mechanism by which Stx is taken up, translocated, and sequestered in EVs was addressed in this study utilizing the B-subunit that binds to the globotriaosylceramide (Gb3) receptor. We found that Stx1B was released in EVs within minutes after stimulation of HeLa cells or red blood cells, detected by live cell imaging and flow cytometry. In the presence of Retro-2.1, an inhibitor of intracellular retrograde trafficking, a continuous release of Stx-positive EVs occurred. EVs from HeLa cells possess the Gb3 receptor on their membrane, and EVs from cells that were treated with a glycosylceramide synthase inhibitor, to reduce Gb3, bound significantly less Stx1B. Stx1B was detected both on the membrane and within the shed EVs. Stx1B was incubated with EVs derived from blood cells, in the absence of cells, and was shown to bind to, and be taken up by, these EVs, as demonstrated by electron microscopy. Using a membrane translocation assay we demonstrated that Stx1B was taken up by blood cell- and HeLa-derived EVs, an effect enhanced by chloropromazine or methyl-ß-cyclodextrin, suggesting toxin transfer within the membrane. This is a novel mechanism by which EVs derived from blood cells can sequester their toxic content, possibly to evade the host response.
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McCluskey, Gavin, Karen E. Morrison, Colette Donaghy, Frederique Rene, William Duddy, and Stephanie Duguez. "Extracellular Vesicles in Amyotrophic Lateral Sclerosis." Life 13, no. 1 (December 31, 2022): 121. http://dx.doi.org/10.3390/life13010121.
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Amyotrophic Lateral Sclerosis is a progressive neurodegenerative disease and is the most common adult motor neuron disease. The disease pathogenesis is complex with the perturbation of multiple pathways proposed, including mitochondrial dysfunction, RNA processing, glutamate excitotoxicity, endoplasmic reticulum stress, protein homeostasis and endosomal transport/extracellular vesicle (EV) secretion. EVs are nanoscopic membrane-bound particles that are released from cells, involved in the intercellular communication of proteins, lipids and genetic material, and there is increasing evidence of their role in ALS. After discussing the biogenesis of EVs, we review their roles in the propagation of pathological proteins in ALS, such as TDP-43, SOD1 and FUS, and their contribution to disease pathology. We also discuss the ALS related genes which are involved in EV formation and vesicular trafficking, before considering the EV protein and RNA dysregulation found in ALS and how these have been investigated as potential biomarkers. Finally, we highlight the potential use of EVs as therapeutic agents in ALS, in particular EVs derived from mesenchymal stem cells and EVs as drug delivery vectors for potential treatment strategies.
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Brunel, Aude, Gaëlle Bégaud, Clément Auger, Stéphanie Durand, Serge Battu, Barbara Bessette, and Mireille Verdier. "Autophagy and Extracellular Vesicles, Connected to rabGTPase Family, Support Aggressiveness in Cancer Stem Cells." Cells 10, no. 6 (May 27, 2021): 1330. http://dx.doi.org/10.3390/cells10061330.
Full textAbstract:
Even though cancers have been widely studied and real advances in therapeutic care have been made in the last few decades, relapses are still frequently observed, often due to therapeutic resistance. Cancer Stem Cells (CSCs) are, in part, responsible for this resistance. They are able to survive harsh conditions such as hypoxia or nutrient deprivation. Autophagy and Extracellular Vesicles (EVs) secretion are cellular processes that help CSC survival. Autophagy is a recycling process and EVs secretion is essential for cell-to-cell communication. Their roles in stemness maintenance have been well described. A common pathway involved in these processes is vesicular trafficking, and subsequently, regulation by Rab GTPases. In this review, we analyze the role played by Rab GTPases in stemness status, either directly or through their regulation of autophagy and EVs secretion.
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Zanetti-Domingues, Laura C., Scott E. Bonner, R. Sumanth Iyer, Marisa L. Martin-Fernandez, and Veronica Huber. "Cooperation and Interplay between EGFR Signalling and Extracellular Vesicle Biogenesis in Cancer." Cells 9, no. 12 (December 8, 2020): 2639. http://dx.doi.org/10.3390/cells9122639.
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Epidermal growth factor receptor (EGFR) takes centre stage in carcinogenesis throughout its entire cellular trafficking odyssey. When loaded in extracellular vesicles (EVs), EGFR is one of the key proteins involved in the transfer of information between parental cancer and bystander cells in the tumour microenvironment. To hijack EVs, EGFR needs to play multiple signalling roles in the life cycle of EVs. The receptor is involved in the biogenesis of specific EV subpopulations, it signals as an active cargo, and it can influence the uptake of EVs by recipient cells. EGFR regulates its own inclusion in EVs through feedback loops during disease progression and in response to challenges such as hypoxia, epithelial-to-mesenchymal transition and drugs. Here, we highlight how the spatiotemporal rules that regulate EGFR intracellular function intersect with and influence different EV biogenesis pathways and discuss key regulatory features and interactions of this interplay. We also elaborate on outstanding questions relating to EGFR-driven EV biogenesis and available methods to explore them. This mechanistic understanding will be key to unravelling the functional consequences of direct anti-EGFR targeted and indirect EGFR-impacting cancer therapies on the secretion of pro-tumoural EVs and on their effects on drug resistance and microenvironment subversion.
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Khanna, Kanika, Sukhmeen Kaur Kohli, Vinod Kumar, Jaspreet Kour, Arun Dev Singh, Tamanna Bhardwaj, Puja Ohri, et al. "Multiple Facets of Plant-Microbiome Associations in Unlocking the Communication Paradigm through Extracellular Vesicles." Current Protein & Peptide Science 22, no. 12 (December 2021): 848–72. http://dx.doi.org/10.2174/1389203722666211109101140.
Full textAbstract:
: Communication among different species across kingdoms occurs through a chain of regulatory molecules that are transferred around cellular boundaries. These molecules are also crucial for defense, virulence, and pathogenesis. In the past, the transport of proteins in long distance communication was observed, but in the present era, the discovery of extracellular vesicles (EVs) has changed our understanding of molecular communication. EVs are not only involved in cell signaling and immunity but also can transfer information by sRNAs, forming a basis for interactions among a wide variety of organisms. Despite extensive research on EVs in other areas, their role in communication between plants and the plant microbiome has been lacking. EVs are potentially involved in protein trafficking along with the transport of lipids and nucleic acids. Interactions between hosts and their microbiomes may also be mediated by EVs, which can be involved in stress responses, immune surveillance and defense, virulence, and signaling, along with many metabolic activities within plant microbiomes. In this review, we have focused on recent information about the role of EVs and the molecules they transport between hosts and microbes. The connection between biofilms and the generation of EVs is also considered. These findings enhance our knowledge about plant-microbiome interactions in terms of immunity and virulence and challenge the conventional viewpoint of inter-kingdom signaling.
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Catani, Lucia, Michele Cavo, and Francesca Palandri. "The Power of Extracellular Vesicles in Myeloproliferative Neoplasms: “Crafting” a Microenvironment That Matters." Cells 10, no. 9 (September 4, 2021): 2316. http://dx.doi.org/10.3390/cells10092316.
Full textAbstract:
Myeloproliferative Neoplasms (MPN) are acquired clonal disorders of the hematopoietic stem cells and include Essential Thrombocythemia, Polycythemia Vera and Myelofibrosis. MPN are characterized by mutations in three driver genes (JAK2, CALR and MPL) and by a state of chronic inflammation. Notably, MPN patients experience increased risk of thrombosis, disease progression, second neoplasia and evolution to acute leukemia. Extracellular vesicles (EVs) are a heterogeneous population of microparticles with a role in cell-cell communication. The EV-mediated cross-talk occurs via the trafficking of bioactive molecules such as nucleic acids, proteins, metabolites and lipids. Growing interest is focused on EVs and their potential impact on the regulation of blood cancers. Overall, EVs have been suggested to orchestrate the complex interplay between tumor cells and the microenvironment with a pivotal role in “education” and “crafting” of the microenvironment by regulating angiogenesis, coagulation, immune escape and drug resistance of tumors. This review is focused on the role of EVs in MPN. Specifically, we will provide an overview of recent findings on the involvement of EVs in MPN pathogenesis and discuss opportunities for their potential application as diagnostic and prognostic biomarkers.
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Wąchalska, Magda, Michał Rychłowski, Kinga Grabowska, Kinga Kowal, Magdalena Narajczyk, Krystyna Bieńkowska-Szewczyk, and Andrea D. Lipińska. "Palmitoylated mNeonGreen Protein as a Tool for Visualization and Uptake Studies of Extracellular Vesicles." Membranes 10, no. 12 (November 27, 2020): 373. http://dx.doi.org/10.3390/membranes10120373.
Full textAbstract:
Extracellular vesicles (EVs) are membranous nanoparticles released by cells as vital mediators of intercellular communication. As such, EVs have become an attractive target for pathogens and cancer cells, which can take control over their cargo composition, as well as their trafficking, shaping the pathogenesis. Despite almost four decades of research on EVs, the number of specific and efficient EV labeling methods is limited, and there is still no universal method for the visualization of their transport in living cells. Lipophilic dyes that non-specifically intercalate into the EVs membranes may diffuse to other membranes, leading to the misinterpretation of the results. Here, we propose a palmitoylated fluorescent mNeonGreen (palmNG) protein as an alternative to chemical dyes for EVs visualization. The Branchiostoma lanceolatum-derived mNeonGreen is a brighter, more stable, and less sensitive to laser-induced bleaching alternative to green fluorescent protein (GFP), which makes it a more potent tag in a variety of fluorescence-based techniques. A palmNG-expressing stable human melanoma cell line was generated using retrovirus gene transfer and cell sorting. This protein partially localizes to cellular membranes, and can be detected inside size-exclusion (SEC)-purified EVs. With the use of flow cytometry and fluorescent confocal microscopy, we performed qualitative and quantitative analyses of palmNG-EVs uptake in recipient human hepatoma cells, in comparison to PKH67-labeled vesicles. Our findings confirm that membrane-embedded mNeonGreen can be successfully applied as a tool in EVs transfer and uptake studies.
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Charreau, Béatrice. "Secretome and Tunneling Nanotubes: A Multilevel Network for Long Range Intercellular Communication between Endothelial Cells and Distant Cells." International Journal of Molecular Sciences 22, no. 15 (July 26, 2021): 7971. http://dx.doi.org/10.3390/ijms22157971.
Full textAbstract:
As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information.
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Maguire, Julie E., Malan Silva, Ken C. Q. Nguyen, Elizabeth Hellen, Andrew D. Kern, David H. Hall, and Maureen M. Barr. "Myristoylated CIL-7 regulates ciliary extracellular vesicle biogenesis." Molecular Biology of the Cell 26, no. 15 (August 2015): 2823–32. http://dx.doi.org/10.1091/mbc.e15-01-0009.
Full textAbstract:
The cilium both releases and binds to extracellular vesicles (EVs). EVs may be used by cells as a form of intercellular communication and mediate a broad range of physiological and pathological processes. The mammalian polycystins (PCs) localize to cilia, as well as to urinary EVs released from renal epithelial cells. PC ciliary trafficking defects may be an underlying cause of autosomal dominant polycystic kidney disease (PKD), and ciliary–EV interactions have been proposed to play a central role in the biology of PKD. In Caenorhabditis elegans and mammals, PC1 and PC2 act in the same genetic pathway, act in a sensory capacity, localize to cilia, and are contained in secreted EVs, suggesting ancient conservation. However, the relationship between cilia and EVs and the mechanisms generating PC-containing EVs remain an enigma. In a forward genetic screen for regulators of C. elegans PKD-2 ciliary localization, we identified CIL-7, a myristoylated protein that regulates EV biogenesis. Loss of CIL-7 results in male mating behavioral defects, excessive accumulation of EVs in the lumen of the cephalic sensory organ, and failure to release PKD-2::GFP-containing EVs to the environment. Fatty acylation, such as myristoylation and palmitoylation, targets proteins to cilia and flagella. The CIL-7 myristoylation motif is essential for CIL-7 function and for targeting CIL-7 to EVs. C. elegans is a powerful model with which to study ciliary EV biogenesis in vivo and identify cis-targeting motifs such as myristoylation that are necessary for EV–cargo association and function.
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Tang, Yunhui, Katie Groom, Larry Chamley, and Qi Chen. "Melatonin, a Potential Therapeutic Agent for Preeclampsia, Reduces the Extrusion of Toxic Extracellular Vesicles from Preeclamptic Placentae." Cells 10, no. 8 (July 27, 2021): 1904. http://dx.doi.org/10.3390/cells10081904.
Full textAbstract:
Preeclampsia, characterised by maternal endothelial cell activation, is triggered by toxic factors, such as placental extracellular vesicles (EVs) from a dysfunctional placenta. The increased oxidative stress seen in the preeclamptic placenta links to endoplasmic reticulum (ER) stress. The ER regulates protein folding and trafficking. When the ER is stressed, proteins are misfolded, and misfolded proteins are toxic. Misfolded proteins can be exported from cells, via EVs which target to other cells where the misfolded proteins may also be toxic. Melatonin is a hormone and antioxidant produced by the pineal gland and placenta. Levels of melatonin are reduced in preeclampsia. In this study we investigated whether melatonin treatment can change the nature of placental EVs that are released from a preeclamptic placenta. EVs were collected from preeclamptic (n = 6) and normotensive (n = 6) placental explants cultured in the presence or absence of melatonin for 18 h. Misfolded proteins were measured using a fluorescent compound, Thioflavin-T (ThT). Endothelial cells were exposed to placental EVs overnight. Endothelial cell activation was measured by the quantification of cell-surface ICAM-1 using a cell-based ELISA. EVs from preeclamptic placentae carried significantly (p < 0.001) more misfolded proteins than normotensive controls. Incubating preeclamptic placental explants in the presence of melatonin (1 µM and 10 µM) significantly (p < 0.001) reduced the misfolded proteins carried by EVs. Culturing endothelial cells in the presence of preeclamptic EVs significantly increased the expression of ICAM-1. This increased ICAM-1 expression was significantly reduced when the endothelial cells were exposed to preeclamptic EVs cultured in the presence of melatonin. This study demonstrates that melatonin reduces the amount of misfolded proteins carried by EVs from preeclamptic placentae and reduces the ability of these EVs to activate endothelial cells. Our study provides further preclinical support for the use of melatonin as a treatment for preeclampsia.
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Savage, John, Ciaran Manus Maguire, and Adrielle Prina-Mello. "Origins to Outcomes: A Role for Extracellular Vesicles in Precision Medicine." Precision Nanomedicine 1, no. 1 (April 19, 2018): 18–42. http://dx.doi.org/10.29016/180419.1.
Full textAbstract:
Extracellular vesicles (EVs) are of great interest in biological research, and though they are a relatively recent discovery, they have rapidly shown great potential for use in clinical applications. The various techniques used in EV isolation along with their respective strengths, weaknesses, and potential for downstream applications are outlined here. A brief description of the different approaches in exosome characterisation are subsequently described. It has been highlighted that despite the recent developments in these processes, there is still a great deal of refinement to be made. EVs are produced by almost all cell types, found in many biological samples, and are implicated in multiple biological processes including cargo trafficking, cell-cell communication, and signal transduction. The presence of these EVs and their varied cargo in a biological sample can be indicative in disease diagnosis, and guide precision medicine-based approaches to disease management. EVs have been reported to actin the benefit of the cell through moderating repair and regeneration, but they can also act to the detriment of the cell through increased tumorigenesis and metastasis. This duality is intriguing as it can allow for the use of EVs in distinct therapeuticapproaches and displays their versatility in potential downstream applications. In this review, examples of the cellular roles of EVs and their applications in pathological and regenerative contexts are explored. In reviewing some of the developments madein recent times, EVs are shown to be very promising both in diagnostic and therapeutic approaches.
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Walsh, Jonathon D., Juan Wang, Molly DeHart, Inna A. Nikonorova, Jagan Srinivasan, and Maureen M. Barr. "Tracking N- and C-termini of C. elegans polycystin-1 reveals their distinct targeting requirements and functions in cilia and extracellular vesicles." PLOS Genetics 18, no. 12 (December 27, 2022): e1010560. http://dx.doi.org/10.1371/journal.pgen.1010560.
Full textAbstract:
The cilium acts as an antenna receiving and sending signals, the latter via extracellular vesicles (EVs). In C. elegans and mammals, the Autosomal Dominant Polycystic Kidney Disease (ADPKD) gene products polycystin-1 (PC1) and polycystin-2 (PC2) localize to both cilia and EVs, act in the same genetic pathway, and function in a sensory capacity, suggesting ancient conservation. However, the functions of the polycystins on cilia and EVs remain enigmatic. We used our C. elegans model and endogenously fluorescent-tagged LOV-1/polycystin-1 to study LOV-1 processing, trafficking, transport, EV biogenesis, and function in living animals. Super resolution, real time imaging reveals that LOV-1 is processed into N-terminal (NTM) and C-terminal (CTM) forms via a conserved GPCR proteolytic site (GPS). The LOV-1 NTM is secreted into the extracellular matrix and not localized to ciliary tip EVs. In contrast, LOV-1 CTM and PKD-2 are co-trafficked, co-transported, and co-localized in cilia and on environmentally released ciliary EVs. LOV-1 CTM requires PKD-2 for ciliary EV localization, while PKD-2 localizes to ciliary EVs independent of LOV-1. We find that LOV-1 but not PKD-2 is required for chemosensation of an ascaroside mating pheromone. These findings indicate that the polycystins LOV-1 and PKD-2 function together and independently and provide insight to how cargo is selected and packaged in ciliary EVs.
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Subramanian, Subbaya, Xianda Zhao, and Ce Yaun. "Tumor exosome mediated immune regulation in colorectal cancer." Journal of Immunology 202, no. 1_Supplement (May 1, 2019): 194.15. http://dx.doi.org/10.4049/jimmunol.202.supp.194.15.
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Abstract Colorectal cancer (CRC) remains the third most common cause of cancer-related deaths in the United States. Most (~85%) of CRC tumors are nonimmunogenic, i.e. they lack a significant number of tumor-infiltrating T cells, and are typically unresponsive to the current immune checkpoint inhibitor-based therapies. T cells isolated from nonimmunogenic, microsatellite stable (MSS) CRC have lower levels of CD28, which provides a costimulatory signal required for T-cell activation, trafficking, proliferation, differentiation, and cytotoxic activity. CRC tumors have elevated levels of miR-503 and its cluster member miR-424, both directly target CD28 in vitro. Our data also showed that these miRNAs are present in extracellular vesicles (EVs) secreted by CRC cells, which are taken up by T cells and affect its proliferation and function in the tumor microenvironment. Further, EVs with these immune suppressive miRNAs support tumor progression in CRC mouse models. Strikingly, in orthotopic models of CRC, mice preconditioned or treated with modified EVs (EVs lacking functional miR-503 and/or miR-424 (Δ503/424-EVs)) are protected against tumor formation and have a significantly reduced tumor burden, while mice receiving control EVs or PBS solution all form tumors. Our results provide preclinical evidence that treating CRC patients with modified EVs (lacking immunosuppressive miRNAs) will significantly improve antitumor immune response and reduce tumor burden. These results provide insights on how cancer cells modulate and suppress the immune response, provide novel targets, and form the basis for a new anti-cancer therapeutic strategy.
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Cerri, Silvia, Cristina Ghezzi, Gerardo Ongari, Stefania Croce, Micol Avenali, Roberta Zangaglia, Donato A. Di Monte, Enza Maria Valente, and Fabio Blandini. "GBA Mutations Influence the Release and Pathological Effects of Small Extracellular Vesicles from Fibroblasts of Patients with Parkinson’s Disease." International Journal of Molecular Sciences 22, no. 4 (February 23, 2021): 2215. http://dx.doi.org/10.3390/ijms22042215.
Full textAbstract:
Heterozygous mutations in the GBA gene, encoding the lysosomal enzyme glucocerebrosidase (GCase), are the strongest known genetic risk factor for Parkinson’s disease (PD). The molecular mechanisms underlying the increased PD risk and the variable phenotypes observed in carriers of different GBA mutations are not yet fully elucidated. Extracellular vesicles (EVs) have gained increasing importance in neurodegenerative diseases since they can vehiculate pathological molecules potentially promoting disease propagation. Accumulating evidence showed that perturbations of the endosomal–lysosomal pathway can affect EV release and composition. Here, we investigate the impact of GCase deficiency on EV release and their effect in recipient cells. EVs were purified by ultracentrifugation from the supernatant of fibroblast cell lines derived from PD patients with or without GBA mutations and quantified by nanoparticle tracking analysis. SH-SY5Y cells over-expressing alpha-synuclein (α-syn) were used to assess the ability of patient-derived small EVs to affect α-syn expression. We observed that defective GCase activity promotes the release of EVs, independently of mutation severity. Moreover, small EVs released from PD fibroblasts carrying severe mutations increased the intra-cellular levels of phosphorylated α-syn. In summary, our work shows that the dysregulation of small EV trafficking and alpha-synuclein mishandling may play a role in GBA-associated PD.
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Sanwald, Julia L., Gereon Poschmann, Kai Stühler, Christian Behrends, Silke Hoffmann, and Dieter Willbold. "The GABARAP Co-Secretome Identified by APEX2-GABARAP Proximity Labelling of Extracellular Vesicles." Cells 9, no. 6 (June 16, 2020): 1468. http://dx.doi.org/10.3390/cells9061468.
Full textAbstract:
The autophagy-related ATG8 protein GABARAP has not only been shown to be involved in the cellular self-degradation process called autophagy but also fulfils functions in intracellular trafficking processes such as receptor transport to the plasma membrane. Notably, available mass spectrometry data suggest that GABARAP is also secreted into extracellular vesicles (EVs). Here, we confirm this finding by the immunoblotting of EVs isolated from cell culture supernatants and human blood serum using specific anti-GABARAP antibodies. To investigate the mechanism by which GABARAP is secreted, we applied proximity labelling, a method for studying the direct environment of a protein of interest in a confined cellular compartment. By expressing an engineered peroxidase (APEX2)-tagged variant of GABARAP—which, like endogenous GABARAP, was present in EVs prepared from HEK293 cells—we demonstrate the applicability of APEX2-based proximity labelling to EVs. The biotinylated protein pool which contains the APEX2-GABARAP co-secretome contained not only known GABARAP interaction partners but also proteins that were found in APEX2-GABARAP’s proximity inside of autophagosomes in an independent study. All in all, we not only introduce a versatile tool for co-secretome analysis in general but also uncover the first details about autophagy-based pathways as possible biogenesis mechanisms of GABARAP-containing EVs.
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Alves, Sara, Joana Pereira, Rupert Mayer, Alexandre Gonçalves, Francis Impens, Didier Cabanes, and Sandra Sousa. "Cells Responding to Closely Related Cholesterol-Dependent Cytolysins Release Extracellular Vesicles with a Common Proteomic Content Including Membrane Repair Proteins." Toxins 15, no. 1 (December 20, 2022): 4. http://dx.doi.org/10.3390/toxins15010004.
Full textAbstract:
The plasma membrane (PM) protects cells from extracellular threats and supports cellular homeostasis. Some pathogens produce pore-forming toxins (PFTs) that disrupt PM integrity by forming transmembrane pores. High PFT concentrations cause massive damage leading to cell death and facilitating infection. Sub-lytic PFT doses activate repair mechanisms to restore PM integrity, support cell survival and limit disease. Shedding of extracellular vesicles (EVs) has been proposed as a key mechanism to eliminate PFT pores and restore PM integrity. We show here that cholesterol-dependent cytolysins (CDCs), a specific family of PFTs, are at least partially eliminated through EVs release, and we hypothesize that proteins important for PM repair might be included in EVs shed by cells during repair. To identify new PM repair proteins, we collected EVs released by cells challenged with sub-lytic doses of two different bacterial CDCs, listeriolysin O and pneumolysin, and determined the EV proteomic repertoire by LC-MS/MS. Intoxicated cells release similar EVs irrespectively of the CDC used. Also, they release more and larger EVs than non-intoxicated cells. A cluster of 70 proteins including calcium-binding proteins, molecular chaperones, cytoskeletal, scaffold and membrane trafficking proteins, was detected enriched in EVs collected from intoxicated cells. While some of these proteins have well-characterized roles in repair, the involvement of others requires further study. As proof of concept, we show here that Copine-1 and Copine-3, proteins abundantly detected in EVs released by intoxicated cells, are required for efficient repair of CDC-induced PM damage. Additionally, we reveal here new proteins potentially involved in PM repair and give new insights into common mechanisms and machinery engaged by cells in response to PM damage.
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Ipinmoroti, Ayodeji O., Brennetta J. Crenshaw, Rachana Pandit, Sanjay Kumar, Brian Sims, and Qiana L. Matthews. "Human Adenovirus Serotype 3 Infection Modulates the Biogenesis and Composition of Lung Cell-Derived Extracellular Vesicles." Journal of Immunology Research 2021 (December 9, 2021): 1–19. http://dx.doi.org/10.1155/2021/2958394.
Full textAbstract:
Adenovirus (Ad) is a major causal agent of acute respiratory infections. However, they are a powerful delivery system for gene therapy and vaccines. Some Ad serotypes antagonize the immune system leading to meningitis, conjunctivitis, gastroenteritis, and/or acute hemorrhagic cystitis. Studies have shown that the release of small, membrane-derived extracellular vesicles (EVs) may offer a mechanism by which viruses can enter cells via receptor-independent entry and how they influence disease pathogenesis and/or host protection considering their existence in almost all bodily fluids. We proposed that Ad3 could alter EV biogenesis, composition, and trafficking and may stimulate various immune responses in vitro. In the present study, we evaluated the impact of in vitro infection with Ad3 vector on EV biogenesis and composition in the human adenocarcinoma lung epithelial cell line A549. Cells were infected in an exosome-free media at different multiplicity of infections (MOIs) and time points. The cell viability was determined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and fluorometric calcein-AM. EVs were isolated via ultracentrifugation. Isolated EV proteins were quantified and evaluated via nanoparticle tracking, transmission electron microscopy, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunoblotting assays. The cell viability significantly decreased with an increase in MOI and incubation time. A significant increase in particle mean sizes, concentrations, and total EV protein content was detected at higher MOIs when compared to uninfected cells (control group). A549 cell-derived EVs revealed the presence of TSG101, tetraspanins CD9 and CD63, and heat shock proteins 70 and 100 with significantly elevated levels of Rab5, 7, and 35 at higher MOIs (300, 750, and 1500) when compared to the controls. Our findings suggested Ad3 could modulate EV biogenesis, composition, and trafficking which could impact infection pathogenesis and disease progression. This study might suggest EVs could be diagnostic and therapeutic advancement to Ad infections and other related viral infections. However, further investigation is warranted to explore the underlying mechanism(s).
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Peruzzotti-Jametti, Luca, Joshua D. Bernstock, Cory M. Willis, Giulia Manferrari, Rebecca Rogall, Erika Fernandez-Vizarra, James C. Williamson, et al. "Neural stem cells traffic functional mitochondria via extracellular vesicles." PLOS Biology 19, no. 4 (April 7, 2021): e3001166. http://dx.doi.org/10.1371/journal.pbio.3001166.
Full textAbstract:
Neural stem cell (NSC) transplantation induces recovery in animal models of central nervous system (CNS) diseases. Although the replacement of lost endogenous cells was originally proposed as the primary healing mechanism of NSC grafts, it is now clear that transplanted NSCs operate via multiple mechanisms, including the horizontal exchange of therapeutic cargoes to host cells via extracellular vesicles (EVs). EVs are membrane particles trafficking nucleic acids, proteins, metabolites and metabolic enzymes, lipids, and entire organelles. However, the function and the contribution of these cargoes to the broad therapeutic effects of NSCs are yet to be fully understood. Mitochondrial dysfunction is an established feature of several inflammatory and degenerative CNS disorders, most of which are potentially treatable with exogenous stem cell therapeutics. Herein, we investigated the hypothesis that NSCs release and traffic functional mitochondria via EVs to restore mitochondrial function in target cells. Untargeted proteomics revealed a significant enrichment of mitochondrial proteins spontaneously released by NSCs in EVs. Morphological and functional analyses confirmed the presence of ultrastructurally intact mitochondria within EVs with conserved membrane potential and respiration. We found that the transfer of these mitochondria from EVs to mtDNA-deficient L929 Rho0 cells rescued mitochondrial function and increased Rho0 cell survival. Furthermore, the incorporation of mitochondria from EVs into inflammatory mononuclear phagocytes restored normal mitochondrial dynamics and cellular metabolism and reduced the expression of pro-inflammatory markers in target cells. When transplanted in an animal model of multiple sclerosis, exogenous NSCs actively transferred mitochondria to mononuclear phagocytes and induced a significant amelioration of clinical deficits. Our data provide the first evidence that NSCs deliver functional mitochondria to target cells via EVs, paving the way for the development of novel (a)cellular approaches aimed at restoring mitochondrial dysfunction not only in multiple sclerosis, but also in degenerative neurological diseases.
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Carvalho, Ana Sofia, Henrique Baeta, Andreia F. A. Henriques, Mostafa Ejtehadifar, Erin M. Tranfield, Ana Laura Sousa, Ana Farinho, et al. "Proteomic Landscape of Extracellular Vesicles for Diffuse Large B-Cell Lymphoma Subtyping." International Journal of Molecular Sciences 22, no. 20 (October 12, 2021): 11004. http://dx.doi.org/10.3390/ijms222011004.
Full textAbstract:
The role of extracellular vesicles (EVs) proteome in diffuse large B-cell lymphoma (DLBCL) pathology, subclassification, and patient screening is unexplored. We analyzed by state-of-the-art mass spectrometry the whole cell and secreted extracellular vesicles (EVs) proteomes of different molecular subtypes of DLBCL, germinal center B cell (GCB subtype), and activated B cell (ABC subtype). After quality control assessment, we compared whole-cell and secreted EVs proteomes of the two cell-of-origin (COO) categories, GCB and ABC subtypes, resulting in 288/1115 significantly differential expressed proteins from the whole-cell proteome and 228/608 proteins from EVs (adjust p-value < 0.05/p-value < 0.05). In our preclinical model system, we demonstrated that the EV proteome and the whole-cell proteome possess the capacity to separate cell lines into ABC and GCB subtypes. KEGG functional analysis and GO enrichment analysis for cellular component, molecular function, and biological process of differential expressed proteins (DEP) between ABC and GCB EVs showed a significant enrichment of pathways involved in immune response function. Other enriched functional categories for DEPs constitute cellular signaling and intracellular trafficking such as B-cell receptor (BCR), Fc_gamma R-mediated phagocytosis, ErbB signaling, and endocytosis. Our results suggest EVs can be explored as a tool for patient diagnosis, follow-up, and disease monitoring. Finally, this study proposes novel drug targets based on highly expressed proteins, for which antitumor drugs are available suggesting potential combinatorial therapies for aggressive forms of DLBCL. Data are available via ProteomeXchange with identifier PXD028267.
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Jewett, Kathryn A., Ruth E. Thomas, Chi Q. Phan, Bernice Lin, Gillian Milstein, Selina Yu, Lisa F. Bettcher, et al. "Glucocerebrosidase reduces the spread of protein aggregation in a Drosophila melanogaster model of neurodegeneration by regulating proteins trafficked by extracellular vesicles." PLOS Genetics 17, no. 2 (February 4, 2021): e1008859. http://dx.doi.org/10.1371/journal.pgen.1008859.
Full textAbstract:
Abnormal protein aggregation within neurons is a key pathologic feature of Parkinson’s disease (PD). The spread of brain protein aggregates is associated with clinical disease progression, but how this occurs remains unclear. Mutations in glucosidase, beta acid 1 (GBA), which encodes glucocerebrosidase (GCase), are the most penetrant common genetic risk factor for PD and dementia with Lewy bodies and associate with faster disease progression. To explore how GBA mutations influence pathogenesis, we previously created a Drosophila model of GBA deficiency (Gba1b) that manifests neurodegeneration and accelerated protein aggregation. Proteomic analysis of Gba1b mutants revealed dysregulation of proteins involved in extracellular vesicle (EV) biology, and we found altered protein composition of EVs from Gba1b mutants. Accordingly, we hypothesized that GBA may influence pathogenic protein aggregate spread via EVs. We found that accumulation of ubiquitinated proteins and Ref(2)P, Drosophila homologue of mammalian p62, were reduced in muscle and brain tissue of Gba1b flies by ectopic expression of wildtype GCase in muscle. Neuronal GCase expression also rescued protein aggregation both cell-autonomously in brain and non-cell-autonomously in muscle. Muscle-specific GBA expression reduced the elevated levels of EV-intrinsic proteins and Ref(2)P found in EVs from Gba1b flies. Perturbing EV biogenesis through neutral sphingomyelinase (nSMase), an enzyme important for EV release and ceramide metabolism, enhanced protein aggregation when knocked down in muscle, but did not modify Gba1b mutant protein aggregation when knocked down in neurons. Lipidomic analysis of nSMase knockdown on ceramide and glucosylceramide levels suggested that Gba1b mutant protein aggregation may depend on relative depletion of specific ceramide species often enriched in EVs. Finally, we identified ectopically expressed GCase within isolated EVs. Together, our findings suggest that GCase deficiency promotes accelerated protein aggregate spread between cells and tissues via dysregulated EVs, and EV-mediated trafficking of GCase may partially account for the reduction in aggregate spread.
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Beer, Katharina B., Jennifer Rivas-Castillo, Kenneth Kuhn, Gholamreza Fazeli, Birgit Karmann, Jeremy F. Nance, Christian Stigloher, and Ann M. Wehman. "Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry." Proceedings of the National Academy of Sciences 115, no. 6 (January 24, 2018): E1127—E1136. http://dx.doi.org/10.1073/pnas.1714085115.
Full textAbstract:
Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans. However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.
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Spinelli, Cristiana, Lata Adnani, Dongsic Choi, and Janusz Rak. "Extracellular Vesicles as Conduits of Non-Coding RNA Emission and Intercellular Transfer in Brain Tumors." Non-Coding RNA 5, no. 1 (December 25, 2018): 1. http://dx.doi.org/10.3390/ncrna5010001.
Full textAbstract:
Non-coding RNA (ncRNA) species have emerged in as molecular fingerprints and regulators of brain tumor pathogenesis and progression. While changes in ncRNA levels have been traditionally regarded as cell intrinsic there is mounting evidence for their extracellular and paracrine function. One of the key mechanisms that enables ncRNA to exit from cells is their selective packaging into extracellular vesicles (EVs), and trafficking in the extracellular space and biofluids. Vesicular export processes reduce intracellular levels of specific ncRNA in EV donor cells while creating a pool of EV-associated ncRNA in the extracellular space and biofluids that enables their uptake by other recipient cells; both aspects have functional consequences. Cancer cells produce several EV subtypes (exosomes, ectosomes), which differ in their ncRNA composition, properties and function. Several RNA biotypes have been identified in the cargo of brain tumor EVs, of which microRNAs are the most studied, but other species (snRNA, YRNA, tRNA, and lncRNA) are often more abundant. Of particular interest is the link between transforming oncogenes and the biogenesis, cargo, uptake and function of tumor-derived EV, including EV content of oncogenic RNA. The ncRNA repertoire of EVs isolated from cerebrospinal fluid and serum is being developed as a liquid biopsy platform in brain tumors.
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Chen, Ding-Wen, Seul Jung, Jian-Meng Fan, Siqi Linsey Zhang, Theresa N. Menna, Zhe Zhang, Sherif Abdelhamed, and Peter Kurre. "Microrna-155 Trafficking Incites Compartmental Inflammation in the Leukemic Niche." Blood 136, Supplement 1 (November 5, 2020): 36. http://dx.doi.org/10.1182/blood-2020-137758.
Full textAbstract:
Compartmental bone marrow (BM) inflammation has been linked to acute myeloid leukemia (AML) progression and hematopoietic dysfunction, yet the underlying mechanisms remain unclear. MicroRNAs (miRs) are capable of broadly deregulating cellular gene expression programs through simultaneous, dose-dependent action on a panel of target genes. MicroRNA-155, a widely known proinflammatory miRNA is overexpressed in AML patients, in particular those with Flt3-ITD. We recently observed that miR-155 is highly abundant in AML patient plasma-derived extracellular vesicles (AML-EV) and identified inflammation as one of the most upregulated gene ontology (GO) categories in a proteomic screen of hematopoietic stem and progenitor cells (HSPCs) exposed to AML-EV. Here, we investigate whether AML-derived miR-155 coordinately regulates inflammatory signaling in the leukemic niche. To better model compartmental inflammation in AML, we first established a highly penetrant, immunocompetent, fully congenic system. Using the monoblastic AML cell line C1498 (CD45.2+), we were able to reliably graft animals without myeloablation, thereby avoiding unintended changes in BM niche function. Across multiple experimental cohorts, we then analyzed the BM plasma secretome and observed dynamic changes in the level of several inflammatory chemokines and cytokines. At sacrifice, flow cytometric analysis of the residual HSPC (CD45.1+) showed concurrent upregulation of key inflammatory response genes (ISG15, STAT1, CXCL10) and evidence of replicative stress (γ-H2AX) in residual HSPC. Consistent with potential trafficking of miR-155 via AML-EV, we next confirmed the enrichment of miR-155 in C1498-EVs, observed gains in miR-155 in BM plasma EVs and demonstrated increased levels in HSPCs following EV uptake. To determine if miR-155 could function as a regulatory hub under inflammatory conditions we utilized four separate target prediction databases (miRDB, TargetScan, miRWalk, and miRanda) to generate miR-155 target gene set. With an initial list of 360 potential gene targets that met a minimum threshold prediction score, we queried six publicly available RNA-Seq datasets to focus on genes belonging to inflammation or DNA damage repair GO groups highly expressed in HSPCs. This yielded a final set of 9 candidate targets (Wee1, E2f2, Rps6ka3, Rps6ka5, Csflr, Mgme1, Rad51, Rad51b, Ercc1). Reasoning that miR-155 target sets are involved in niche inflammation and genotoxicity in AML, these computationally predicted targets will undergo testing and validation for involvement in inflammatory and genotoxic stress. Chronic inflammation in the AML niche contributes to disease persistence and failure of residual hematopoiesis. Our study will determine the role of miR-155 as a regulatory hub for compartmental inflammatory signaling, and simultaneously serve as a potential opportunity for novel therapeutic target discovery in leukemic BM. Disclosures No relevant conflicts of interest to declare.
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Ouweneel, Amber B., Michael J. Thomas, and Mary G. Sorci-Thomas. "The ins and outs of lipid rafts: functions in intracellular cholesterol homeostasis, microparticles, and cell membranes." Journal of Lipid Research 61, no. 5 (December 30, 2019): 676–86. http://dx.doi.org/10.1194/jlr.tr119000383.
Full textAbstract:
Cellular membranes are not homogenous mixtures of proteins; rather, they are segregated into microdomains on the basis of preferential association between specific lipids and proteins. These microdomains, called lipid rafts, are well known for their role in receptor signaling on the plasma membrane (PM) and are essential to such cellular functions as signal transduction and spatial organization of the PM. A number of disease states, including atherosclerosis and other cardiovascular disorders, may be caused by dysfunctional maintenance of lipid rafts. Lipid rafts do not occur only in the PM but also have been found in intracellular membranes and extracellular vesicles (EVs). Here, we focus on discussing newly discovered functions of lipid rafts and microdomains in intracellular membranes, including lipid and protein trafficking from the ER, Golgi bodies, and endosomes to the PM, and we examine lipid raft involvement in the production and composition of EVs. Because lipid rafts are small and transient, visualization remains challenging. Future work with advanced techniques will continue to expand our knowledge about the roles of lipid rafts in cellular functioning.
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Lovisa, Federica, Anna Garbin, Sara Crotti, Piero Di Battista, Ilaria Gallingani, Carlotta Caterina Damanti, Anna Tosato, et al. "Increased Tenascin C, Osteopontin and HSP90 Levels in Plasmatic Small Extracellular Vesicles of Pediatric ALK-Positive Anaplastic Large Cell Lymphoma: New Prognostic Biomarkers?" Diagnostics 11, no. 2 (February 6, 2021): 253. http://dx.doi.org/10.3390/diagnostics11020253.
Full textAbstract:
Over the past 15 years, several biological and pathological characteristics proved their significance in pediatric anaplastic lymphoma kinase (ALK)-positive anaplastic large-cell lymphoma (ALCL) prognostic stratification. However, the identification of new non-invasive disease biomarkers, relying on the most important disease mechanisms, is still necessary. In recent years, plasmatic circulating small extracellular vesicles (S-EVs) gathered great importance both as stable biomarker carriers and active players in tumorigenesis. In the present work, we performed a comprehensive study on the proteomic composition of plasmatic S-EVs of pediatric ALCL patients compared to healthy donors (HDs). By using a mass spectrometry-based proteomics approach, we identified 50 proteins significantly overrepresented in S-EVs of ALCL patients. Gene Ontology enrichment analysis disclosed cellular components and molecular functions connected with S-EV origin and vesicular trafficking, whereas cell adhesion, glycosaminoglycan metabolic process, extracellular matrix organization, collagen fibril organization and acute phase response were the most enriched biological processes. Of importance, consistently with the presence of nucleophosmin (NPM)-ALK fusion protein in ALCL cells, a topological enrichment analysis based on Reactome- and Kyoto Encyclopedia of Genes and Genomes (KEGG)-derived networks highlighted a dramatic increase in proteins of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway in ALCL S-EVs, which included heat shock protein 90-kDa isoform alpha 1 (HSP90AA1), osteopontin (SPP1/OPN) and tenascin C (TNC). These results were validated by Western blotting analysis on a panel of ALCL and HD cases. Further research is warranted to better define the role of these S-EV proteins as diagnostic and, possibly, prognostic parameters at diagnosis and for ALCL disease monitoring.
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Lopera-Vasquez, R., M. Hamdi, V. Maillo, C. Nunez, M. Yanez-Mo, M. A. Ramirez, A. Gutierrez-Adan, P. Bermejo-Alvarez, and D. Rizos. "99 EXTRACELLULAR VESICLES OF BOVINE OVIDUCTAL FLUID MODIFY THE GENE EXPRESSION ON BOVINE IN VITRO-DERIVED EMBRYOS." Reproduction, Fertility and Development 28, no. 2 (2016): 179. http://dx.doi.org/10.1071/rdv28n2ab99.
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Extracellular vesicles (EVs) act as intercellular communicators through their protein, lipid, and mRNA content. The interaction of EVs from oviducal environment and the first stages of embryo development is currently an enigma. The aim of the present study was to evaluate the developmental competence and the expression profile of bovine blastocysts cultured with previously purified EVs recovered from ampullary and isthmic oviducal fluid (OF) under different centrifugal forces. OF-EVs recovered from oviducts of slaughtered heifers in early luteal phase were quantified with a nanoparticle tracking analysis system, and their integrity and size were assessed by electron microscopy. In vitro-produced zygotes were cultured in SOF+3 mg mL–1 BSA (C–), C– with 3 × 105 OF-EVs/mL from the ampulla (A) and isthmus (I) isolated at 1 × 103 (A10k and I10k, respectively) and 1 × 105 (A100k and I100k, respectively) × g. A control culture group of SOF+5% FCS (C+) was included. Blastocyst development was recorded on Day 7, 8, and 9 (D0: day of fertilization). Blastocysts on Days 7/8 cultured in C–, C+, I10k, and I100k were used to measure the relative mRNA expression of genes related with membrane trafficking (AQP3, AQP11, and ATP1A1), metabolism (LDLR and LDHA), and epigenetics (DNMT3A, IGF2R, GRB10, and SNRPN) by RT-qPCR. One-way ANOVA was used for statistical analysis. The size of ampullary and isthmic OF-EVs was similar with a mean of 220 nm. The concentration of I10k was significantly lower compared with A100k (3.6 × 108 v. 10.5 × 108 EVs/mL, respectively; P < 0.05); however, no differences were found in the rest of the groups with a mean concentration of 7.6 × 108 EVs/mL. EVs and C– groups showed a delayed embryo development at Day 7 compared with C+ (range: 12.0–13.8 v. 20.6%, respectively, P < 0.05); however, it was compensated at Days 8 and 9 (Day 9 range: 28.5–30.8%). The water channel related protein AQP3, associated with blastocoel formation, water, and cryoprotectant movement during cryopreservation, was up-regulated in I10k and I100k blastocysts compared with C+. The lipid receptor LDLR, proposed as a regulator of lipid uptake in blastocysts, was significantly down-regulated in C+ compared with the other groups, a possible consequence of a higher concentration of lipids in the C+ group. The de novo DNA methyltransferase DNMT3A and the imprinting gene SNRPN were down-regulated in the C+ compared with I100k, suggesting alterations in imprinting. In conclusion, bovine isthmic OF-EVs supplementation in in vitro embryo culture has a positive effect on gene expression patterns of developmental related genes compared with serum supplementation, suggesting an association between the oviducal environment and the developing embryo. Funded by the Spanish Ministry of Science and Innovation (AGL2012–37510 and AGL2012–39652-C02–01).
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Muse, Oluwatoyosi, Rushad Patell, Christian Peters, Sol Schulman, Emale Darzi, Calvin Schuster, Ling Huang, et al. "The Unfolded Protein Response Causes Prothrombotic Transformation of Pancreatic Cancer Linking Tumor Progression with Cancer-Associated Thrombosis." Blood 134, Supplement_1 (November 13, 2019): 632. http://dx.doi.org/10.1182/blood-2019-123544.
Full textAbstract:
Thrombosis is a common complication of advanced stage cancer. Yet the underlying mechanisms that link tumor progression to clot formation are poorly understood. The unfolded protein response (UPR) is associated with malignant transformation in pancreatic cancer, but whether or not activation of the UPR is linked to cancer thrombosis has not previously been evaluated. To determine whether UPR signaling functions in the prothrombotic transformation of pancreatic cancer, we exposed pancreatic adenocarcinoma cells (HPAF-II cells) to three UPR inducers (tunicamycin, triptolide or thapsigargin) that act via independent mechanisms. Induction of UPR resulted in the release of thrombogenic material into the supernatant as evidenced by a ~3-fold increase in thrombin generation in pelleted material. Release of thrombogenic material was inhibited by siRNA-mediated knockdown of UPR components including IRE1a (80±3% decrease) or PERK (60±10% decrease). Chemical inhibition of UPR also inhibited release of thrombogenic material from HPAF-II cells. Exposure to the IRE1a inhibitor, MKC-3946, resulted in a 70±10% decrease in thrombin generation in pelleted material, and incubation with the PERK inhibitor, GSK2606414, caused an 80±5% decrease. Characterization of the thrombogenic activity revealed that it was present on extracellular vesicles (EVs) and was inhibited by anti-TF antibodies. Flow cytometry demonstrated on average a 3-fold increase in fluorescence of TF-bearing EVs following UPR induction. Electron microscopy showed that the HPAF II EVs ranged from 100-500 mm and demonstrated increased clustering following UPR induction. Three-color immunofluorescence microscopy of HPAF II cells with labeling of actin, nuclei, and TF showed that induction of the UPR resulted in actin-poor membrane blebs rich in TF. Apoptosis as detected by caspase-3 cleavage was not observed under these conditions. Brefeldin A, which inhibits vesicular transport between the endoplasmic reticulum and the Golgi, inhibited UPR-induced generation of TF-bearing EVs, indicating that UPR-mediated vesicular trafficking contributes to TF-bearing EV formation. To evaluate the possibility of an association between the UPR and cancer thrombosis in the clinical setting, we analyzed plasmas collected from pancreatic cancer patients who were monitored prospectively for the development of venous thromboembolism (including a lower extremity ultrasound performed at baseline and at 2 months). Proteomic analysis was performed using Somalogic technology to evaluate ~1300 analytes in plasmas from nine pancreatic cancer patients who subsequently developed venous thromboembolism and ten patients with similar pancreatic cancer characteristics who remained free of venous thromboembolism. Evaluation of eight UPR markers present in the SOMAscan panel demonstrated significant upregulation in plasmas of patients who developed clots compared to those who did not (p = 0.0001). Our current findings support a model whereby activation of the UPR results in increased vesicular trafficking leading to the release of TF-bearing EVs. These observations indicate a mechanistic link between tumor progression in pancreatic cancer and cancer associated thrombosis. Disclosures Peters: Platelet BioGenesis: Employment. Zwicker:Portola: Consultancy; Parexel: Consultancy; Bayer: Consultancy; Seattle Genetics: Consultancy; Daiichi: Consultancy; Quercegen: Research Funding; Incyte: Research Funding. Flaumenhaft:Relay Therapeutics: Consultancy; PlateletDiagnostics: Consultancy, Other: Founder.
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Ramachandran, Sowmya, Amit K. Verma, Kapil Dev, Yamini Goyal, Deepti Bhatt, Mohammed A. Alsahli, Arshad Husain Rahmani, et al. "Role of Cytokines and Chemokines in NSCLC Immune Navigation and Proliferation." Oxidative Medicine and Cellular Longevity 2021 (July 16, 2021): 1–20. http://dx.doi.org/10.1155/2021/5563746.
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With over a million deaths every year around the world, lung cancer is found to be the most recurrent cancer among all types. Nonsmall cell lung carcinoma (NSCLC) amounts to about 85% of the entire cases. The other 15% owes it to small cell lung carcinoma (SCLC). Despite decades of research, the prognosis for NSCLC patients is poorly understood with treatment options limited. First, this article emphasises on the part that tumour microenvironment (TME) and its constituents play in lung cancer progression. This review also highlights the inflammatory (pro- or anti-) roles of different cytokines (ILs, TGF-β, and TNF-α) and chemokine (CC, CXC, C, and CX3C) families in the lung TME, provoking tumour growth and subsequent metastasis. The write-up also pinpoints recent developments in the field of chemokine biology. Additionally, it covers the role of extracellular vesicles (EVs), as alternate carriers of cytokines and chemokines. This allows the cytokines/chemokines to modulate the EVs for their secretion, trafficking, and aid in cancer proliferation. In the end, this review also stresses on the role of these factors as prognostic biomarkers for lung immunotherapy, apart from focusing on inflammatory actions of these chemoattractants.
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Cheerathodi, Mujeeb, Dingani Nkosi, Allaura S. Cone, Sara B. York, and David G. Meckes. "Epstein-Barr Virus LMP1 Modulates the CD63 Interactome." Viruses 13, no. 4 (April 15, 2021): 675. http://dx.doi.org/10.3390/v13040675.
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Tetraspanin CD63 is a cluster of cell surface proteins with four transmembrane domains; it is associated with tetraspanin-enriched microdomains and typically localizes to late endosomes and lysosomes. CD63 plays an important role in the cellular trafficking of different proteins, EV cargo sorting, and vesicle formation. We have previously shown that CD63 is important in LMP1 trafficking to EVs, and this also affects LMP1-mediated intracellular signaling including MAPK/ERK, NF-κB, and mTOR activation. Using the BioID method combined with mass spectrometry, we sought to define the broad CD63 interactome and how LMP1 modulates this network of interacting proteins. We identified a total of 1600 total proteins as a network of proximal interacting proteins to CD63. Biological process enrichment analysis revealed significant involvement in signal transduction, cell communication, protein metabolism, and transportation. The CD63-only interactome was enriched in Rab GTPases, SNARE proteins, and sorting nexins, while adding LMP1 into the interactome increased the presence of signaling and ribosomal proteins. Our results showed that LMP1 alters the CD63 interactome, shifting the network of protein enrichment from protein localization and vesicle-mediated transportation to metabolic processes and translation. We also show that LMP1 interacts with mTOR, Nedd4 L, and PP2A, indicating the formation of a multiprotein complex with CD63, thereby potentially regulating LMP1-dependent mTOR signaling. Collectively, the comprehensive analysis of CD63 proximal interacting proteins provides insights into the network of partners required for endocytic trafficking and extracellular vesicle cargo sorting, formation, and secretion.
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Picca, Anna, Flora Guerra, Riccardo Calvani, Cecilia Bucci, Maria Rita Lo Monaco, Anna Rita Bentivoglio, Francesco Landi, Roberto Bernabei, and Emanuele Marzetti. "Mitochondrial-Derived Vesicles as Candidate Biomarkers in Parkinson’s Disease: Rationale, Design and Methods of the EXosomes in PArkiNson Disease (EXPAND) Study." International Journal of Molecular Sciences 20, no. 10 (May 14, 2019): 2373. http://dx.doi.org/10.3390/ijms20102373.
Full textAbstract:
The progressive loss of dopaminergic neurons in the nigro-striatal system is a major trait of Parkinson’s disease (PD), manifesting clinically as motor and non-motor symptoms. Mitochondrial dysfunction and oxidative stress are alleged pathogenic mechanisms underlying aggregation of misfolded α-synuclein that in turn triggers dopaminergic neurotoxicity. Peripheral processes, including inflammation, may precede and contribute to neurodegeneration. Whether mitochondrial dyshomeostasis in the central nervous system and systemic inflammation are linked to one another in PD is presently unclear. Extracellular vesicles (EVs) are delivery systems through which cells can communicate or unload noxious materials. EV trafficking also participates in mitochondrial quality control (MQC) by generating mitochondrial-derived vesicles to dispose damaged organelles. Disruption of MQC coupled with abnormal EV secretion may play a role in the pathogenesis of PD. Furthermore, due to its bacterial ancestry, circulating mitochondrial DNA can elicit an inflammatory response. Therefore, purification and characterisation of molecules packaged in, and secreted through, small EVs (sEVs)/exosomes in body fluids may provide meaningful insights into the association between mitochondrial dysfunction and systemic inflammation in PD. The EXosomes in PArkiNson Disease (EXPAND) study was designed to characterise the cargo of sEVs/exosomes isolated from the serum of PD patients and to identify candidate biomarkers for PD.