Exosome Shedding Research Articles

Exosome Shedding Is Concordant with Objective Treatment Response Rate and Stratifies Time to Progression in Treatment Naïve, Non-Resectable Hepatocellular Carcinoma

Translational strategies to characterize and monitor extracellular vesicles such as exosome (EX) shedding and the clinical impact of this data within hepatocellular carcinoma (HCC) remains unclear. In this study, EX shedding was assessed in early-stage HCC and evaluated as a stratification factor for time to progression (TTP) following first-cycle liver-directed therapy (LDT). Plasma EXs were isolated from HCC patients undergoing LDT using ultracentrifugation. Purified EXs were stained using markers CD9 and CD63 and quantified using an ImageStreamX flow cytometer. Circulating EXs expressing CD9 were isolated at 10-fold higher levels compared to CD63. The intensity of CD9+ EX shedding following LDT was positively correlated with treatment response. High post-LDT CD9+ EX shedding stratified TTP risk with a 30% lower frequency of disease progression at 1 year following LDT. Post-LDT high CD9+ EX shedding was observed in 100% (10/10) of patients successfully bridged to liver transplantation while only 22% (2/9) of patients with tumor progression had high CD9+ EX shedding post-LDT. CD9+ EX shedding also stratified TTP risk within the first cycle objective response rate (ORR) group, identifying patients still at higher disease progression. EX shedding was concordant with imaging response rate, stratified TTP in early-stage HCC, and may have important implications for assessing post-LDT viable, biologically aggressive HCC.

Experimental Validation of Novel Glypican 3 Exosomes for the Detection of Hepatocellular Carcinoma in Liver Cirrhosis.

Background and AimsHepatocellular carcinoma (HCC) developing in the context of preexisting cirrhosis is characterized by impaired autophagy that results in increased exosome release. This study was conducted to determine whether circulating exosomes expressing glypican 3 (GPC3) could be utilized as a biomarker for HCC detection and treatment response in patients with cirrhosis.MethodsImmunohistochemistry was performed to assess p62 and GPC3 expression in the lesion and adjacent tissue from cirrhosis with HCC. GPC3-enriched exosomes were captured by an enzyme-linked immunosorbent assay (ELISA). The diagnostic specificity of serum exosome-derived GPC3 (eGPC3) was determined using samples obtained from malignancy-free controls, malignancy-free cirrhotics, cirrhotics with confirmed HCC, and patients with a non-HCC malignancy. The performance of eGPC3 was validated using serum samples of HCC patients received chemotherapy.ResultsWe found that the expression of p62 and GPC3 was significantly increased in HCC tissues compared to adjacent cirrhotic liver. Impaired autophagy and exosome shedding were confirmed in HCC cell lines. Mass spectroscopic analysis revealed that GPC3 was enriched in exosomes isolated from HCC cell lines. An affinity ELISA assay was developed that specifically captures GPC3 positive exosomes in the serum. Total exosome concentration and eGPC3 were significantly elevated in cirrhotic patients with HCC as compared to the reference control groups. Furthermore, decreases in post-treatment exosome concentration and eGPC3 levels were more closely correlated with response to locoregional chemotherapy compared to change in serum AFP in HCC patients awaiting liver transplantation.ConclusionWe developed an affinity exosome capture assay to detect GPC3 enriched exosomes. Our preliminary assessment shows that GPC3 positive exosomes can be used for HCC detection and prediction of treatment outcomes.

A High-Fat Diet Attenuates AMPK α1 in Adipocytes to Induce Exosome Shedding and Nonalcoholic Fatty Liver Development In Vivo.

Exosomes are important for intercellular communication, but the role of exosomes in the communication between adipose tissue (AT) and the liver remains unknown. The aim of this study is to determine the contribution of AT-derived exosomes in nonalcoholic fatty liver disease (NAFLD). Exosome components, liver fat content, and liver function were monitored in AT in mice fed a high-fat diet (HFD) or treated with metformin or GW4869 and with AMPKα1-floxed (Prkaα1fl/fl/wild-type [WT]), Prkaα1−/−, liver tissue-specific Prkaα1−/−, or AT-specific Prkaα1−/− modification. In cultured adipocytes and white AT, the absence of AMPKα1 increased exosome release and exosomal proteins by elevating tumor susceptibility gene 101 (TSG101)–mediated exosome biogenesis. In adipocytes treated with palmitic acid, TSG101 facilitated scavenger receptor class B (CD36) sorting into exosomes. CD36-containing exosomes were then endocytosed by hepatocytes to induce lipid accumulation and inflammation. Consistently, an HFD induced more severe lipid accumulation and cell death in Prkaα1−/− and AT-specific Prkaα1−/− mice than in WT and liver-specific Prkaα1−/− mice. AMPK activation by metformin reduced adipocyte-mediated exosome release and mitigated fatty liver development in WT and liver-specific Prkaα1−/− mice. Moreover, administration of the exosome inhibitor GW4869 blocked exosome secretion and alleviated HFD-induced fatty livers in Prkaα1−/− and adipocyte-specific Prkaα1−/− mice. We conclude that HFD-mediated AMPKα1 inhibition promotes NAFLD by increasing numbers of AT CD36-containing exosomes.

Genetic Association of Clinical Complications for Genes Differentially Expressed in Reticulocytes of Sickle Cell Anemia

Introduction Sickle cell anemia (SCA) is characterized by hemolysis, a tissue hypoxia-driven increase in erythropoietin production, and a marked reticulocytosis reflecting stress erythropoiesis. Reticulocytes in SCA constitute 4-15% or more of peripheral red blood cells. Sickle reticulocytes express adhesion molecules that are normally cleared by exosome shedding, including α4β1 integrin, which is thought to contribute to vaso-occlusive crises by binding to vascular endothelial cell vascular cell adhesion molecule 1. Our recent study of the reticulocyte transcriptome in SCA versus healthy African Americans, using RNA-Seq, revealed differential genes that were implicated in a broad range of biological processes including terminal erythroid differentiation, the unfolded protein response, and altered membrane systems. In this study, we assessed the genetic association of these differential genes with disease complications relevant to sickle reticulocytes. Methods We analyzed vaso-occlusive complications including episodic severe pain and chronic avascular necrosis. We also analyzed hemolysis and anemia measured by a hemolysis index and hemoglobin concentration, respectively. The patient cohort combined 525 adolescent and adult patients from Walk PHaSST and 377 pediatric patients from PUSH. Genomic DNA isolated from peripheral blood mononuclear cells of Walk-PHaSST and PUSH patients were genotyped using Illumina Human 610-Quad SNP array and the genotypes were phased and imputed to 1000 genomes phase 3 data. Single nucleotide polymorphisms (SNPs) with minor allele frequency (MAF) >0.01 and imputation r2>0.7 were tested for gene-wise genetic association using SKAT. For severe pain and avascular necrosis, the covariates included age, gender, β globin genotype severity (HHbSS and HbSβ0-thalassemia versus HbSC and HbSβ+-thalassemia), and population stratification. For hemolysis and hemoglobin concentration, the covariates included age, gender, β globin genotype severity, alpha thalassemia, hydroxyurea treatment, recent blood transfusion, and population stratification. Results A total of 111 genes were analyzed. These genes were classified into 13 functional categories based on published research (transcription, post-transcription, translation, histone modification, cell cycle arrest, mitophagy, metabolism, iron homeostasis, antioxidative system, unfolded protein response, membrane skeleton and structural proteins, membrane adhesion molecules and blood group antigens, and membrane transporters). For each functional class, the number of significant gene-wise associations the functional class contains was compared to the number of significant associations from the other classes combined. Defining significance of gene-wise association at nominal P=0.05, we found that the "Unfolded protein response" contained more significant gene-wise associations with severe pain than the other functional classes (one-sided Fisher's exact test P=0.046), driven by genes FUCA1 (alpha-L- fucosidase) and CLN8 (associated with endoplasmic reticulum function). "Membrane skeleton and structural proteins" showed a trend of enrichment for hemolysis (P=0.057), driven by genes MYO5B and CD2AP, and for avascular necrosis (P=0.075), driven by genes MYLK, CAVIN2 and PALLD. Defining the significance of gene-wise association at a less stringent nominal P=0.1, "membrane adhesion molecules and blood group antigens" showed a trend of enrichment in association with severe pain (P=0.097), driven by genes GYPA, ITGA4 and ITGB1, the latter two encoding α4β1 integrin subunits which are abnormally present on the reticulocyte membrane and have been implicated in sickle vaso-occlusion. Regional scans revealed the SNPs with the most significant associations within these genes. Discussion Our results reveal enrichment of genetic association with vaso-occlusive and hemolytic complications in gene functional categories that showed altered expression in sickle reticulocytes. Future study is needed to validate these associations. Disclosures Gordeuk: CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding; Ironwood: Research Funding; Novartis: Consultancy.

MicroRNA-34a-mediated death of acute myeloid leukemia stem cells through apoptosis induction and exosome shedding inhibition via histone deacetylase 2 targeting.

We investigated the role of leukemia stem cells in chemoresistance and recurrence of acute myeloid leukemia. Total RNA was isolated from cells or tissues using TRIzol reagent. Cell viability was assessed with the tetrazolium assay. MicroRNA-34a (miR-34a), which acts on cell death regulation pathways, was noticeably downregulated in non-M3 acute myeloid leukemia stem cells compared with normal hematopoietic stem cells. Furthermore, inhibition of miR-34a-mediated suppression in leukemia stem cells was associated with poor clinical outcomes and impaired treatment efficacy in acute myeloid leukemia. Transfection with a miR-34a mimic triggered leukemia stem cell death and prevented leukemia. Bioinformatics analysis and a dual-luciferase reporter assay showed that miR-34a targeted the 3'-untranslated region of histone deacetylase 2, and the reinforced expression of miR-34a remarkably stimulated the expression of histone deacetylase 2 in leukemia stem cells. Ectopic miR-34a expression triggered death of leukemia stem cells via pathways involving the Janus kinase 1-signal transducer and activator of transcription 2-p53 axis. Targeting leukemia stem cells to trigger cell death through upregulation of miR-34a expression could be used to diagnose and treat acute myeloid leukemia.

MiR-139 Controls Viability Of Ovarian Cancer Cells Through Apoptosis Induction And Exosome Shedding Inhibition By Targeting ATP7A.

BackgroundEmerging proof suggests that microRNA (miRNA) malfunction is correlated to the generation and development of multiple malignancies. It has been proven that miRNA (miR)-139 represses a variety of malignancies. However, the understanding of its impact on ovarian cancer (OC) is insufficient. We aimed to investigate the expression and function of miR-139 in OC.MethodsThe expression of mir-139 was detected and recorded, and the relationship of mir-139 remaining OC cells was explored. At the same time, we studied the correlation between ATP7A and mir-139 by the luciferase reporter test, Western blot, and quantitative real-time reverse transcription PCR.ResultsThe expression of miR-139 was remarkably downregulated in OC specimens. Furthermore, excessive miR-139 expression noticeably inhibited the migration, colony generation, proliferation, and invasion of OC cells. In addition, excessive miR-139 expression remarkably repressed the death and the expression of proteins related to cell death in OC cells, as well as inhibited the shedding of exosomes. According to the luciferase reporter test, Western blot, and quantitative real-time reverse transcription PCR, miR-139 directly targeted ATP7A. Furthermore, the expression of ATP7A was found to be negatively related to miR-139 levels in OC specimens. It was revealed via a rescue experiment that excessive ATP7A expression counteracted the repressive effect of miR-139 in OC cells.ConclusionIt was revealed via an in vivo study that miR-139 remarkably inhibited the growth of malignancies by downregulating ATP7A in nude mice. miR-139 represses the development of malignancies in OC by directly targeting ATP7A, offering an innovative approach for molecular therapy of OC.

Causes and Consequences of the Dysregulated Maternal Renin-Angiotensin System in Preeclampsia.

A healthy pregnancy outcome depends on the activation of the renin-angiotensin-aldosterone system (RAAS) as a regulated, integrated response to the growing demands of the conceptus. Both the circulating RAAS and the intrarenal renin-angiotensin system (iRAS) play major roles in cardiovascular function and fluid and electrolyte homeostasis. The circulating RAAS becomes dysfunctional in preeclampsia and we propose that dysregulation of the iRAS plays a role in development of the clinical syndrome known as preeclampsia. Experimental studies in animals have shown that placental renin, when released into the maternal circulation, can cause hypertension. We postulate that abnormal placental development is associated with over-secretion of renin and other RAS proteins/angiotensin (Ang) peptides by the placenta/decidua into the maternal circulation. We hypothesise that this is because of increased shedding of exosomes and other placental particles into the maternal circulation that not only contain RAS proteins and peptides but also microRNAs (miRNAs) that target RAS mRNAs, and Ang II type 1 receptor autoantibodies (AT1R-AAs), that are agonists for, and have the same actions as, Ang II. As a result, there is both suppression of the circulating RAAS that is responsible for maintaining maternal homeostasis and activation of the iRAS. Together with altered vascular reactivity to Ang peptides, the iRAS causes hypertension, renal damage and secondary changes in the neurohumoral control of the maternal circulation and fluid and electrolyte balance, which contribute to the pathophysiology of preeclampsia.

MiR-34c-5p promotes eradication of acute myeloid leukemia stem cells by inducing senescence through selective RAB27B targeting to inhibit exosome shedding.

Leukemia stem cells (LSCs) are responsible for acute myeloid leukemia (AML) chemotherapy resistance and relapse. Here, we discovered that miR-34c-5p, a microRNA central to the senescence regulation network, was significantly down-regulated in AML (non-acute promyelocytic leukemia, non-APL) stem cells compared to that in normal hematopoietic stem cells (HSCs). The lower expression of miR-34c-5p in LSCs was closely correlated to the adverse prognosis and poor responses to therapy of AML patients. Increased miR-34c-5p expression induced LSCs senescence ex vivo, prevented leukemia development and promoted the eradication of LSCs in immune deficient mice. Mechanistically, forced expression of miR-34-5p induced senescence in LSCs through p53-p21Cip1-Cyclin-dependent kinase (CDK)/Cyclin or p53-independent CDK/Cyclin pathways. Exosome-mediated transfer of miR-34c-5p was one of the reasons for miR-34c-5p deficiency in LSCs. Furthermore, miR-34c-5p could increase its intracellular level by inhibiting exosome-mediated transfer via a positive feedback loop through RAB27B, a molecule that promotes exosome shedding. Overall, this study establishes a new strategy for treatment of AML patients by targeting LSCs to reinitiate senescence via increased miR-34c-5p expression. This miRNA-mediated tumor stem cell senescence could also have important therapeutic value in other malignancies.

Characterization of the ultrastructure in the uterovaginal junction of the hen

In poultry, the infundibulum is the place of fertilization, eggshell production, and sperm storage, while its uterovaginal junction (UVJ) is regarded as the most important site, which has abundant sperm storage tubules (SST). We examined the ultrastructure of the epithelium with relation to its unique secretory cytology in the UVJ of hens using transmission electron microscopy. The epithelium of the UVJ is lined with ciliated and secretory cells. Ciliated cells are characterized with light and dense secretory granules in supernuclear cytoplasm. Dense secretory granules in ciliated cells are larger in diameter (oneμm), surrounded with a transparent rim and concentric layers, whereas the dense granules in the ciliated cells of SST are smaller (0.52μm) in size and not surrounded by any transparent rim or layer. Ciliated cells also are involved in the shedding of exosomes and secretory vesicles in the lumen. Secretory exosomes are in close contact with cilia and directly release from the apical border into the lumen. Cell junctions are widely distributed between these cells. The secretory cells are associated with the release of secretions via apocrine blebs from microvilli and secretory vesicles, which protrude out from the surface of the epithelium. The dense secretory granules in these cells are smaller in size (0.6μm), absent of a transparent rim or layers, and are released into the lumen through secretory vesicles. The intracellular multivesicular body (MVB) also is observed in the supernuclear cytoplasm of secretory cells, which are related to the production of exosomes. In general, the apical protrusion of the epithelium in the form of apocrine secretions, the releasing of exosomes, the identification of intracellular MVB, and the release of dense granules give the epithelium a distinct morphology in the UVJ of the hen oviduct.

Enterovirus 71 transmission by exosomes establishes a productive infection in human neuroblastoma cells.

Exosomes are small secreted cellular vesicles for intercellular communications which contain proteins, mRNAs, and microRNAs (miRNAs). Recent studies have shown that exosomes play an important role in the transmission of infectious agents including hepatitis C virus, human immunodeficiency virus, and so on. However, the role of exosomes in the transfer of enterovirus 71 (EV71) between host cells remains unknown. In this study, we show that the exosomes derived from EV71-infected rhabdomyosarcoma cells contain EV71 RNA and capsid protein VP1, determined by quantitative reverse transcription-PCR (QRT-PCR) and Western blot analysis. The shedding of exosomes containing virus can establish a productive infection in human neuroblastoma cell line (SK-N-SH). A comparative analysis of neutralization by EV71-specific immunoglobulins showed different levels of neutralization of exosomes-mediated infection compared with free virus. In conclusion, exosomes from EV71-infected cells may play an important role in virus dissemination and are partially resisted to antibody neutralization. Our results suggest that there is an exosomal route of EV71 transmission infection.

Exosomes mediate embryo and maternal interactions at implantation and during pregnancy.

Shedding of exosomes and microvesicles is now a well-recognized, important method of cell-cell communication in a number of different cell types. However, their importance in the female reproductive tract and in mediating embryo-maternal interactions during pregnancy has only recently been recognized. Here we review the current literature as to release of extracellular vesicles by uterine cells, the embryo,, and placental trophoblast cells; how release is regulated; and the different types of signaling molecules and genetic information contained within such vesicles. We also discuss the role of these exosomes and microvesicles in regulating critical processes during implantation and pregnancy such as angiogenesis, matrix remodeling, alterations in immune function and pathological effects in gestational diseases. A better understanding of the role of exosomes and microvesicles in reproduction may lead to the development of new therapeutic approaches for treatment of infertility and pregnancy complications.

Abstract 1658: Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment

Abstract The gene encoding the cytoskeletal regulator DIAPH3 is lost at high frequency in metastatic prostate cancer, and silencing of DIAPH3 evokes a transition to an amoeboid phenotype in multiple tumor cell backgrounds. This amoeboid transformation is accompanied by increases in plasma membrane deformations (blebbing), cell migration, invasion, and metastasis. DIAPH3 silencing also promotes the shedding of atypically large (>1μm) extracellular vesicles (EV) containing bioactive cargo. Whether loss of DIAPH3 also stimulates the release of bioactive nano-sized EV (exosomes) is not established. Here we examined the mechanism of release and potential biological functions of EV shed from DIAPH3-silenced and other prostate cancer cells. We observed that stimulation of LNCaP cells with the prostate stroma-derived growth factor heparin-binding EGF-like growth factor (HB-EGF), combined with p38MAPK inhibition caused exosome shedding, a process mediated by ERK1/2 hyperactivation. DIAPH3 silencing in DU145 cells also increased rates of exosome production. EV isolated from DIAPH3-silenced cells activated AKT1 and androgen signaling, increased proliferation of recipient tumor cells, and suppressed proliferation of human macrophages and peripheral blood mononuclear cells (PBMC). DU145-derived EV contained miR-125a, which suppressed AKT1 expression and proliferation in recipient human peripheral blood mononuclear cells and macrophages. Our findings suggest that EV across a wide size range (∼70nm to >1um) are produced by amoeboid prostate cancer cells as a result of DIAPH3 loss and/or growth factor stimulation. These EV may condition the tumor microenvironment through multiple mechanisms, including promoting the growth of cancer cells and suppression of tumor-infiltrating immune cells. Citation Format: Jayoung Kim, Samantha Morley, Minh Le, Denis Bedoret, Dale Umetsu, Dolores Di Vizio, Michael Freeman. Enhanced shedding of extracellular vesicles from amoeboid prostate cancer cells: Potential effects on the tumor microenvironment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1658. doi:10.1158/1538-7445.AM2014-1658

Umbilical Cord Stem Cells Induce Glial Fate in Neural Progenitor Cells

s / Placenta 35 (2014) A1eA112 A93 Objectives: The Wharton’s jelly (WJ) of the umbilical cord is derived from the mesoderm of the primitive streak which invades the connecting stalk and primary chorionic villi. Mesenchymal stem cells in the mesoderm differentiate and participate in neo-vasculogenesis of the umbilical and placental circulation. Whether these cells have a continued role in placental/umbilical vascular growth and maturity is not known. Fetal stem cells have been shown to traffic and repair injured myocardial endothelium in mice (Kara et al. 2012). Their ability to interact with the uterine endothelium has not been investigated. The aim of this study was to investigate whether umbilical stem cells (WJMSC) can interact and influence umbilical vein and uterine endothelial monolayers. Methods: Human umbilical vein (HUVEC) and uterine microvascular endothelial cells (HUtMEC) were grown to 70% confluence in endothelial media and to full confluence in mixed media (endothelial: stem cell media). Isolated WJMSC, labelled with the fluorescent tracker, PKH26 was added on top. Cell-cell interactions were monitored with fluorescence time-lapse microscopy. Key events were analysed by confocal microscopy after vascular endothelial (VE) cadherin immunocytochemistry. Results: WJMSC displayed non-apoptotic blebbing, amoeboid movement and shedding of exosomes as early events. There was a time lag of 60 min before transmigration of WJMSC via the paracellular pathway to subendothelial positions. This was concomitant with disruption of VE-cadherin (statistically significant reduction of continuous junctional profiles). By 16h, a majority of WJMSC were resident underneath the endothelial monolayers; junctional profiles returned to normal values. At 22h, the cocultures showed a higher % of paracellular clefts with continuous VEcadherin labelling, indicative of increased junctional maturity. Conclusion: Our data suggests that umbilical mesenchymal stem cells can interact with and influence junctional organisation of both fetal and uterine endothelial cells. Their role, if any, in uterine vascular re-modelling during pregnancy requires investigation. P2.102-N. UMBILICAL CORD STEM CELLS INDUCE GLIAL FATE IN NEURAL PROGENITOR CELLS Byron Oppliger , Marianne J€ orger-Messerli , Camilla Marini , Ursula Reinhart , Daniel V. Surbek , Andreina Schoeberlein a Department of Obstetrics and Gynecology, University Hospital, Department of Clinical Research, University of Bern, Bern, Bern, Switzerland; Graduate School for Cellular and Biomedical Sciences (GCB), University of Bern, Bern, Bern,

Cell-to-cell transfer of M. tuberculosis antigens optimizes CD4 T cell priming.

During Mycobacterium tuberculosis and other respiratory infections, optimal T cell activation requires pathogen transport from the lung to a local draining lymph node (LN). However, the infected inflammatory monocyte-derived dendritic cells (DCs) that transport M. tuberculosis to the local lymph node are relatively inefficient at activating CD4 T cells, possibly due to bacterial inhibition of antigen presentation. We found that infected migratory DCs release M. tuberculosis antigens as soluble, unprocessed proteins for uptake and presentation by uninfected resident lymph node DCs. This transfer of bacterial proteins from migratory to local DCs results in optimal priming of antigen-specific CD4 T cells, which are essential in controlling tuberculosis. Additionally, this mechanism does not involve transfer of the whole bacterium and is distinct from apoptosis or exosome shedding. These findings reveal a mechanism that bypasses pathogen inhibition of antigen presentation by infected cells and generates CD4 T cell responses that control the infection.

Release of heat shock proteins (Hsps) and the effects of extracellular Hsps on neural cells and tissues

Hsps were long thought to be cytoplasmic proteins with functions restricted to the intra-cellular compartment. However, an increasing number of observations show that they can be released into the extra-cellular space and can then have a wide variety of affects on other cells. The mechanism of release remains unknown, but may involve the shedding of exosomes, which are vesicles containing cytoplasmic constituents. In the nervous system, it appears that glial cells can release the 70 kD Hsps and that neurons exposed to extra-cellular Hsps are more likely to survive injury than their naïve counterparts. These phenomena have great potential significance in the development of neuroprotective therapeutic strategies utilizing the heat shock protein response.