Increased Extracellular Vesicles Release Research Articles

Distinct role of Sirtuin 1 (SIRT1) and Sirtuin 2 (SIRT2) in inhibiting cargo-loading and release of extracellular vesicles

Exosomes, vehicles for intercellular communication, are formed intracellularly within multivesicular bodies (MVBs) and are released upon fusion with the plasma membrane. For their biogenesis, proper cargo loading to exosomes and vesicle traffic for extracellular release are required. Previously we showed that the L-type lectin, LMAN2, limits trans-Golgi Network (TGN)-to-endosomes traffic of GPRC5B, an exosome cargo protein, for exosome release. Here, we identified that the protein deacetylase sirtuin 2 (SIRT2) as a novel interactor of LMAN2. Loss of SIRT2 expression resulted in exosomal release of LMAN2, a Golgi resident protein, along with increased exosomal release of GPRC5B. Furthermore, knockout of SIRT2 increased total number of extracellular vesicles (EVs), indicating increased MVB-to-EV flux. While knockout of SIRT1 increased EV release with enlarged late endolysosome, knockout of SIRT2 did not exhibit endolysosome enlargement for increased EV release. Taken together, our study suggests that SIRT2 regulates cargo loading to MVBs and MVB-to-EV flux through a mechanism distinct from that of SIRT1.

A cell-based assay for CD63-containing extracellular vesicles.

Extracellular vesicles (EVs) are thought to be important in cell-cell communication and have elicited extraordinary interest as potential biomarkers of disease. However, quantitative methods to enable elucidation of mechanisms underlying release are few. Here, we describe a cell-based assay for monitoring EV release using the EV-enriched tetraspanin CD63 fused to the small, ATP-independent reporter enzyme, Nanoluciferase. Release of CD63-containing EVs from stably expressing cell lines was monitored by comparing luciferase activity in culture media to that remaining in cells. HEK293, U2OS, U87 and SKMel28 cells released 0.3%-0.6% of total cellular CD63 in the form of EVs over 5 hrs, varying by cell line. To identify cellular machinery important for secretion of CD63-containing EVs, we performed a screen of biologically active chemicals in HEK293 cells. While a majority of compounds did not significantly affect EV release, treating cells with the plecomacrolides bafilomycin or concanamycin, known to inhibit the V-ATPase, dramatically increased EV release. Interestingly, alkalization of the endosomal lumen using weak bases had no effect, suggesting a pH-independent enhancement of EV release by V-ATPase inhibitors. The ability to quantify EVs in small samples will enable future detailed studies of release kinetics as well as further chemical and genetic screening to define pathways involved in EV secretion.

EP4 Antagonist-Elicited Extracellular Vesicles from Mesenchymal Stem Cells Rescue Cognition/Learning Deficiencies by Restoring Brain Cellular Functions.

Adult brains have limited regenerative capacity. Consequently, both brain damage and neurodegenerative diseases often cause functional impairment for patients. Mesenchymal stem cells (MSCs), one type of adult stem cells, can be isolated from various adult tissues. MSCs have been used in clinical trials to treat human diseases and the therapeutic potentials of the MSC‐derived secretome and extracellular vesicles (EVs) have been under investigation. We found that blocking the prostaglandin E2/prostaglandin E2 receptor 4 (PGE2/EP4) signaling pathway in MSCs with EP4 antagonists increased EV release and promoted the sorting of specific proteins, including anti‐inflammatory cytokines and factors that modify astrocyte function, blood–brain barrier integrity, and microglial migration into the damaged hippocampus, into the EVs. Systemic administration of EP4 antagonist‐elicited MSC EVs repaired deficiencies of cognition, learning and memory, inhibited reactive astrogliosis, attenuated extensive inflammation, reduced microglial infiltration into the damaged hippocampus, and increased blood–brain barrier integrity when administered to mice following hippocampal damage. stem cells translational medicine 2019

Oncogenic H-Ras Expression Induces Fatty Acid Profile Changes in Human Fibroblasts and Extracellular Vesicles.

Extracellular vesicles (EVs) are lipid bilayer surrounded particles that are considered an additional way to transmit signals outside the cell. Lipids have not only a structural role in the organization of EVs membrane bilayer, but they also represent a source of lipid mediators that may act on target cells. Senescent cells are characterized by a permanent arrest of cell proliferation, but they are still metabolically active and influence nearby tissue secreting specific signaling mediators, including those carried by EVs. Notably, cellular senescence is associated with increased EVs release. Here, we used gas chromatography coupled to mass spectrometry to investigate the total fatty acid content of EVs released by fibroblasts undergoing H-RasV12-induced senescence and their parental cells. We find that H-RasV12 fibroblasts show increased level of monounsaturated and decreased level of saturated fatty acids, as compared to control cells. These changes are associated with transcriptional up-regulation of specific fatty acid-metabolizing enzymes. The EVs released by both controls and senescent fibroblasts show a higher level of saturated and polyunsaturated species, as compared to parental cells. Considering that fibroblasts undergoing H-RasV12-induced senescence release a higher number of EVs, these findings indicate that senescent cells release via EVs a higher amount of fatty acids, and in particular of polyunsaturated and saturated fatty acids, as compared to control cells.

Palmitic Acid Induces Extracellular Vesicle Release in Human Renal Proximal Tubule Epithelial Cells

Lipotoxicity, an accumulation of intracellular lipid metabolites, has been proposed as an important pathogenic mechanism leading to kidney dysfunction in the context of metabolic disease. Palmitate, a predominant lipid derivative, can cause lipoapoptosis and the release of inflammatory extracellular vesicles (EVs) in hepatocytes. This study was aimed to investigate whether free fatty acid palmitate could increase EV release in renal tubular epithelial cells. A human proximal tubule epithelial cell line, HK‐2, was incubated with 1% bovine serum albumin (BSA), BSA‐conjugated palmitic acid (PA, 250 μM), BSA‐conjugated oleic acid (OA, 250 μM), or diluent (control) for 24 hours. The released EVs were isolated by ultracentrifugation, then characterized and quantified by immunoblot and nanoparticle‐tracking analysis (NTA). To examine the uptake of EVs by tubular cells, isolated EVs were further labelled and applied to HK‐2 cells. According to the NTA, the size distribution of HK‐2‐derived EVs was 30–200 nm with a similar mode size in all four groups. Interestingly, incubation of HK‐2 cells with 1%BSA alone significantly increased the release of EVs compared to the vehicle control. Moreover, BSA‐induced EV release was further increased by approximately two‐fold in the presence of PA, whereas EV production was not significantly changed by the addition of OA. PA (250 μM)‐enhanced EV release in HK‐2 is independent of cell apoptosis since apoptosis‐related proteins were not different between BSA and BSA‐PA groups. Both confocal microscopy and flow cytometry analyses confirmed that labeled lipid‐induced EVs were taken up by HK‐2 cells. Collectively, our results indicate that saturated fatty acid palmitate stimulates EV release from HK‐2 cells. Lipid‐induced EVs may serve as an untapped source to evaluate novel signaling pathways contributing to nephropathy progression.Support or Funding InformationNIH Diabetes and Digestive Kidney Disease Grant Number: R25DK1136559MSM Interdisciplinary Sleep/Clinical Cardiovascular Research Training Program (T32)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.

Extracellular vesicles (EVs) are nanoscale membrane-derived vesicles that serve as intercellular messengers carrying lipids, proteins, and genetic material. Substantial evidence has shown that cancer-derived EVs, secreted by tumor cells into the blood and other bodily fluids, play a critical role in modulating the tumor microenvironment and affecting the pathogenesis of cancer. Here we demonstrate for the first time that squamous cell carcinoma (SCC) EVs were enriched with the C-terminal fragment of desmoglein 2 (Dsg2), a desmosomal cadherin often overexpressed in malignancies. Overexpression of Dsg2 increased EV release and mitogenic content including epidermal growth factor receptor and c-Src. Inhibiting ectodomain shedding of Dsg2 with the matrix metalloproteinase inhibitor GM6001 resulted in accumulation of full-length Dsg2 in EVs and reduced EV release. When cocultured with Dsg2/green fluorescence protein-expressing SCC cells, green fluorescence protein signal was detected by fluorescence-activated cell sorting analysis in the CD90+ fibroblasts. Furthermore, SCC EVs activated Erk1/2 and Akt signaling and enhanced fibroblast cell proliferation. In vivo, Dsg2 was highly up-regulated in the head and neck SCCs, and EVs isolated from sera of patients with SCC were enriched in Dsg2 C-terminal fragment and epidermal growth factor receptor. This study defines a mechanism by which Dsg2 expression in cancer cells can modulate the tumor microenvironment, a step critical for tumor progression.-Overmiller, A. M., Pierluissi, J. A., Wermuth, P. J., Sauma, S., Martinez-Outschoorn, U., Tuluc, M., Luginbuhl, A., Curry, J., Harshyne, L. A., Wahl, J. K. III, South, A. P., Mahoney, M. G. Desmoglein 2 modulates extracellular vesicle release from squamous cell carcinoma keratinocytes.

MP65-01 EXTRACELLULAR VESICLES AS A POSSIBLE MECHANISM FOR BACILLUS CALMETTE-GUÉRIN IMMUNOTHERAPY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III1 Apr 2017MP65-01 EXTRACELLULAR VESICLES AS A POSSIBLE MECHANISM FOR BACILLUS CALMETTE-GUÉRIN IMMUNOTHERAPY Carlos Ortiz-Bonilla, Christopher Silvers, Peng-Nien Yin, Edward Messing, and Yi-Fen Lee Carlos Ortiz-BonillaCarlos Ortiz-Bonilla More articles by this author , Christopher SilversChristopher Silvers More articles by this author , Peng-Nien YinPeng-Nien Yin More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1995AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Intravesical Bacillus Calmette-Guérin (BCG) immunotherapy has been used to treat non-muscle invasive bladder cancer (BC) for nearly 40 years but its underlying mechanism remains largely unknown. It is generally believed that BCG adheres to integrin ?5?1 in the urothelial lining by interacting with fibronectin, then triggers an immune response cascade. Extracellular vesicles (EVs), small membrane-bound vesicles, act as immune modulators by transferring molecular cargos to recipient cells. We hypothesize that EVs derived from BC cells play key roles in mediating BCG-induced anti-tumor host immune responses, and the patient derived EVs may serve as predictive biomarkers that can differentiate BCG responders from non-responders. METHODS BC cell lines, human T24 and murine MB49, and immortalized bladder SV-HUC cells were treated with 1-4x106 CFU/ml live BCG. After 12-72 hours, secreted EVs were isolated by serial ultracentrifugation and analyzed by Nanoparticle Tracking Analysis. Cell lysate and total RNA was collected for immuno-molecular profiling by quantitative PCR and Western blotting analyses. The alterations in EV secretion, gene and protein expression, and molecules from BC derived EVs in response to BCG were compared. Urinary EVs were collected and purified before and after BCG patients' 1st and 3rd BCG instillations and EV secretion profiles were compared. RESULTS In response to BCG, SV-HUC cells showed decreased EV secretion and their immuno- molecules were significantly reduced compared with EVs from naïve SV-HUC cells. In contrast, in BC cells the EV secretion rate was significantly induced by BCG as well as the expression of the key molecules in modulating immune response, such as MHC and co-stimulatory molecules at gene and protein levels. Critically, we found that BC cells, but not SV-HUC cells, released immuno-molecules containing EVs in response to BCG. Importantly, we found that urinary EV numbers were increased significantly after the 3rd BCG instillation in BCG responders, but not in BCG-non-responders in a pilot study. CONCLUSIONS We conclude that BCG treatment resulted in increased EV release from BC cells as well as in increased EVs in urine of BC patients. In addition, BCG induced expression of key immuno-modulatory molecules in BC cells and within the EVs. This up-regulated expression of immuno-molecules in response to BCG supports the hypothesis that EVs have a role in activating the immune system during BCG immunotherapy. The immunologically active EVs detected in patients' urine can be further explored as predictive biomarkers. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e852-e853 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Carlos Ortiz-Bonilla More articles by this author Christopher Silvers More articles by this author Peng-Nien Yin More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Activated Stat5 trafficking Via Endothelial Cell-derived Extracellular Vesicles Controls IL-3 Pro-angiogenic Paracrine Action.

Soluble factors and cell-derived extracellular vesicles (EVs) control vascular cell fate during inflammation. The present study investigates the impact of Interleukin 3 (IL-3) on EV release by endothelial cells (ECs), the mechanisms involved in EV release and paracrine actions. We found that IL-3 increases EV release, which is prevented by IL-3Ralpha blockade. EVs released upon IL-3 stimulation were able to induce pro-angiogenic signals as shown by chromatin immunoprecipitation (ChIP) assay performed on the promoter region of cyclin D1 and tridimensional tube-like structure formation. We herein demonstrate that these effects rely on the transfer of miR-126-3p, pre-miR-126 and, more importantly, of activated signal transduction and activator of transcription 5 (pSTAT5) from IL-3-EV cargo into recipient ECs. We show, using the dominant negative form (ΔN)STAT5 and an activated STAT5 (1*6STAT5) constructs, that STAT5 drives IL-3-mediated EV release, miR-126-3p and pSTAT5 content. Finally, using EVs recovered from ΔNSTAT5 expressing ECs, we provide evidence that miR-126-3p and pSTAT5 trafficking is relevant for IL-3-mediated paracrine pro-angiogenic signals. These results indicate that IL-3 regulates EC-EV release, cargo and IL-3 angiogenic paracrine action via STAT5. Moreover, these results provide evidence that EC-derived IL-3-EVs can serve as pro-angiogenic clinical delivery wound healing devices.

Alcohol stimulates macrophage activation through caspase-dependent hepatocyte derived release of CD40L containing extracellular vesicles

The mechanisms by which hepatocyte exposure to alcohol activates inflammatory cells such as macrophages in alcoholic liver disease (ALD) are unclear. The role of released nano-sized membrane vesicles, termed extracellular vesicles (EV), in cell-to-cell communication has become increasingly recognized. We tested the hypothesis that hepatocytes exposed to alcohol may increase EV release to elicit macrophage activation. Primary hepatocytes or HepG2 hepatocyte cell lines overexpressing ethanol-metabolizing enzymes alcohol dehydrogenase (HepG2(ADH)) or cytochrome P450 2E1 (HepG2(Cyp2E1)) were treated with ethanol and EV release was quantified with nanoparticle tracking analysis. EV mediated macrophage activation was monitored by analysing inflammatory cytokines and macrophage associated mRNA expression, immunohistochemistry, biochemical serum alanine aminotransferase and triglycerides analysis in our in vitro macrophage activation and in vivo murine ethanol feeding studies. Ethanol significantly increased EV release by 3.3-fold from HepG2(Cyp2E1) cells and was associated with activation of caspase-3. Blockade of caspase activation with pharmacological or genetic approaches abrogated alcohol-induced EV release. EV stimulated macrophage activation and inflammatory cytokine induction. An unbiased microarray-based approach and antibody neutralization experiments demonstrated a critical role of CD40 ligand (CD40L) in EV mediated macrophage activation. In vivo, wild-type mice receiving a pan-caspase, Rho kinase inhibitor or with genetic deletion of CD40 (CD40(-/-)) or the caspase-activating TRAIL receptor (TR(-/-)), were protected from alcohol-induced injury and associated macrophage infiltration. Moreover, serum from patients with alcoholic hepatitis showed increased levels of CD40L enriched EV. In conclusion, hepatocytes release CD40L containing EV in a caspase-dependent manner in response to alcohol exposure which promotes macrophage activation, contributing to inflammation in ALD.