Membrane Microvesicles Research Articles

Characterizing the immunoprofile and endogenous immune response to squamous cell carcinomas of the head and neck to guide development of effective immunotherapy strategies

Squamous cell carcinoma of the head and neck (HNSCC) is the 6th leading cause of cancer by incidence worldwide with approximately 600,000 new cases per year. Unfortunately, only 40-50% of these patients will survive for 5 years. In order to study the immune response in patients with this disease we have developed a HNSCC tumor bank to compliment our Oral, Head and Neck Cancer Program. This tumor bank is cryopreserving enzymatically isolated viable cells from resected tumors (n = 128). We are also attempting to develop primary cell lines (10 lines established) and are isolating and assessing tumor-specific function of tumor-infiltrating lymphocytes (TIL) (n = 49). Since HNSCC can express immune inhibitory molecules and secrete suppressive factors, we are developing single stains and multi-spectral imaging protocols to assess the immune contexture of the tumor microenvironment by immunohistochemistry and immunoflouresence and are overlaying these studies with T cell functional activity and ultimately, with clinical outcome. Preliminary analyses suggest that tumor-specific T cells can be detected in 68% (N = 16) of patients evaluated. A goal of these studies is to identify strategies that will allow tailoring of therapy for patients with HNSCC. One component is to identify which inhibitors are present in a given tumor. Since not every tumor appears to contain TIL capable of recognizing autologous tumor, strategies to prime tumor-specific T cells represents another area of interest. DPV-001 is a microvesicle vaccine, DRibbles, that contains an average of at least 66 proteins that are overexpressed by HNSCC (TCGA provisional RNASeq n = 303 pts). The vaccine also contains multiple DAMPs and agonist activity for TLR 2, 3, 4, 7 and 9 packed into stable double membrane microvesicles that are targeted to CLEC9A+ APC. To increase potential activity against HPV positive cancers we have developed a mosaic construct encoding E6 and E7 peptides for a number of HPV strains and are evaluating both protein and gene-based HPV vaccine strategies. We are using the HNSCC TIL lines to evaluate DRibble vaccines and potential for adoptive immunotherapy trials. Support: NCI U43CA165048 and R44 CA121612 (TLH), OMS (HH, RBB), Steve and Cindy Harder, Robert W. and Elsie Franz, Wes and Nancy Lematta, Lynn and Jack Loacker, and The Chiles foundation (BAF).

Erythrophagocytosis of lead-exposed erythrocytes by renal tubular cells: possible role in lead-induced nephrotoxicity.

Background: Nephrotoxicity associated with lead poisoning has been frequently reported in epidemiological studies, but the underlying mechanisms have not been fully described.Objectives: We examined the role of erythrocytes, one of the major lead reservoirs, in lead-associated nephrotoxicity.Methods and results: Co-incubation of lead-exposed human erythrocytes with HK-2 human renal proximal tubular cells resulted in renal tubular cytotoxicity, suggesting a role of erythrocytes in lead-induced nephrotoxicity. Morphological and flow cytometric analyses revealed that HK-2 cells actively phagocytized lead-exposed erythrocytes, which was associated with phosphatidylserine (PS) externalization on the erythrocyte membrane and generation of PS-bearing microvesicles. Increased oxidative stress and up-regulation of nephrotoxic biomarkers, such as NGAL, were observed in HK-2 cells undergoing erythrophagocytosis. Moreover, TGF-β, a marker of fibrosis, was also significantly up-regulated. We examined the significance of erythrophagocytosis in lead-induced nephrotoxicity in rats exposed to lead via drinking water for 12 weeks. We observed iron deposition and generation of oxidative stress in renal tissues of lead-exposed rats, as well as the histopathological alterations such as tubulointerstitial lesions, fibrosis, and up-regulation of KIM-1, NGAL, and TGF-β.Conclusions: Our data strongly suggest that erythrophagocytosis and subsequent iron deposition in renal tubular cells could significantly enhance nephrotoxicity following lead exposure, providing insight on lead-associated kidney damages.Citation: Kwon SY, Bae ON, Noh JY, Kim K, Kang S, Shin YJ, Lim KM, Chung JH. 2015. Erythrophagocytosis of lead-exposed erythrocytes by renal tubular cells: possible role in lead-induced nephrotoxicity. Environ Health Perspect 123:120–127; http://dx.doi.org/10.1289/ehp.1408094

The interleukin (IL)-31/IL-31R axis contributes to tumor growth in human follicular lymphoma.

Interleukin (IL)-31A binds to an heterodimer composed of IL-31 receptor A (IL-31RA) and Oncostatin M Receptor (OSMR). The IL-31/IL-31R complex is involved in the pathogenesis of various skin diseases, including cutaneous T-cell lymphoma. No information is available on the relations between the IL-31/IL-31R complex and B-cell lymphoma. Here we have addressed this issue in follicular lymphoma (FL), a prototypic germinal center(GC)-derived B-cell malignancy. IL-31 enhanced primary FL cell proliferation through IL-31R-driven signal transducer and activator of transcription factor 1/3 (STAT1/3), extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt phosphorylation. In contrast, GC B cells did not signal to IL-31 in spite of IL-31R expression. GC B cells expressed predominantly the inhibitory short IL-31RA isoform, whereas FL cells expressed predominantly the long signaling isoform. Moreover, GC B cells lacked expression of other IL-31RA isoforms potentially involved in the signaling pathway. IL-31 protein expression was significantly higher in surface membrane than in cytosol of both FL and GC B cells. IL-31 was detected in plasma membrane microvesicles from both cell types but not released in soluble form in culture supernatants. IL-31 and IL-31RA expression was higher in lymph nodes from FL patients with grade IIIa compared with grade I/II, suggesting a paracrine and/or autocrine role of IL-31/IL-31RA complex in tumor progression through microvesicle shedding.

Gamma-glutamyltransferases: exploring the complexity of a multi-functional family of enzymes

Gamma-glutamyltransferases: exploring the complexity of a multi-functional family of enzymes

Paclitaxel is incorporated by mesenchymal stromal cells and released in exosomes that inhibit in vitro tumor growth: a new approach for drug delivery.

Mesenchymal stromal cells (MSCs) have been proposed for delivering anticancer agents because of their ability to home in on tumor microenvironment. We found that MSCs can acquire strong anti-tumor activity after priming with Paclitaxel (PTX) through their capacity to uptake and then release the drug. Because MSCs secrete a high amount of membrane microvesicles (MVs), we here investigated the role of MVs in the releasing mechanism of PTX. The murine SR4987 line was used as MSC model. The release of PTX from SR4987 in the conditioned medium (CM) was checked by HPLC and the anti-tumor activity of both CM and MVs was tested on the human pancreatic cell line CFPAC-1. MVs were isolated by ultracentrifugation, analyzed by transmission (TEM) and scanning electron microscopy (SEM), and the presence of PTX by the Fourier transformed infrared (FTIR) microspectroscopy. SR4987 loaded with PTX (SR4987PTX) secreted a significant amount of PTX and their CM possessed strong anti-proliferative activity on CFPAC-1. At TEM and SEM, SR4987PTX showed an increased number of "vacuole-like" structures and shed a relevant number of MVs, but did not differ from untreated SR4987. However, SR4987PTX-derived-MVs (SR4987PTX-MVs) demonstrated a strong anti proliferative activity on CFPAC-1. FTIR analysis of SR4987PTX-MVs showed the presence of an absorption spectrum in the corresponding regions of the PTX marker, absent in MVs from SR4987. Our work is the first demonstration that MSCs are able to package and deliver active drugs through their MVs, suggesting the possibility of using MSCs as a factory to develop drugs with a higher cell-target specificity.

THU0473 Deep Sequencing Reveals Differential Small RNA Expression in Serum Exosomes from Children with Juvenile Idiopathic Arthritis

BackgroundJuvenile idiopathic arthritis (JIA) is the most common rheumatic disease of childhood. The aetiology of JIA remains largely unknown. The number of effective medications for the treatment of RA has...

Exosomes and microvesicles: identification and targeting by particle size and lipid chemical probes.

Exosomes and microvesicles are two classes of submicroscopic vesicle released by cells into the extracellular space. Collectively referred to as extracellular vesicles, these membrane containers facilitate important cell-cell communication by carrying a diverse array of signaling molecules, including nucleic acids, proteins, and lipids. Recently, the role of extracellular vesicle signaling in cancer progression has become a topic of significant interest. Methods to detect and target exosomes and microvesicles are needed to realize applications of extracellular vesicles as biomarkers and, perhaps, therapeutic targets. Detection of exosomes and microvesicles is a complex problem as they are both submicroscopic and of heterogeneous cellular origins. In this Minireview, we highlight the basic biology of extracellular vesicles, and address available biochemical and biophysical detection methods. Detectible characteristics described here include lipid and protein composition, and physical properties such as the vesicle membrane shape and diffusion coefficient. In particular, we propose that detection of exosome and microvesicle membrane curvature with lipid chemical probes that sense membrane shape is a distinctly promising method for identifying and targeting these vesicles.

A176: Deep Sequencing Reveals Differential Small RNA Expression in Serum Exosomes of Children With Juvenile Idiopathic Arthritis

Background/Purpose:The number of effective medications for the treatment of JIA has rapidly expanded, and studies have demonstrated that aggressive treatment of early JIA results in improved clinical outcomes. Therefore biomarkers for early diagnosis and prediction of therapeutic outcome are highly desirable. Exosomes, endosome‐derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell‐cell communication. Of these RNA transcripts, microRNAs are the most abundant. These microRNAs have great potential as disease biomarkers. This study was aimed at characterizing exosomal microRNA profiles in children with polyarticular JIA and determining whether such profiles might be disease state‐specific.Methods:Plasma samples were obtained from children with RF‐negative, polyarticular JIA as well as healthy controls (HC; n = 5). Children with JIA had either active, untreated disease (AD, n = 5) or were in clinical remission on medication (CRM; n = 5) as established by the Wallace criteria. Exosomes were isolated from plasma using ExoQuick Precipitation methods. Expression of CD63 (a marker for exosomes) in all samples was validated by western blot. MicroRNA samples were isolated from exosomes using the miRNeasy Micro kit. Small RNA libraries were prepared using standard methods. Sequencing was performed using the Illumina HiSeq 2500 platform. The pass filter reads were mapped to the genome (GRCH37/hg19) using Bowtie 2. Differentially expressed (DE) microRNA were determined using edgeR using a P‐value of 0.05.Results:Plasma exosomal RNAs demonstrated both disease and disease‐state specificity. For example, when we compared AD to HC, there were 30 DE miRNAs (24 down‐regulated, 6 up‐regulated) and 15 small nucleolar RNA (24 down‐regulated, 6 up‐regulated) that demonstrated a 1.6 fold change or greater. We found 11 DE miRNAs (down‐regulated) and 35 DE snoRNA (up‐regulated) that showed a 2.0 fold of greater difference when we compared CRM vs HC. Ingenuity pathway analysis demonstrated that the DE miRNAs are associated with inflammatory disease, connective tissue disorders and skeletal and muscular disorders (e.g. miR‐10, miR‐22, miR‐29, miR‐146, miR‐154, miR‐320, miR‐374 and miR‐500). Four miRNAs (miR‐146a, miR‐146b, miR‐24‐2 and miR‐320b‐2) were common to the AD vs HC and the CRM vs HC comparisons. Further evidence that the plasma exosomal RNA repertoire is disease‐state specific was seen in direct comparison of the AD vs CRM samples. There were 21 DE miRNAs (2 down‐regulated, 19 up‐regulated) and 24 DE snoRNA in the AD vs CRM comparison. These miRNA included let‐7, miR‐21, miR‐143, miR‐148, miR‐154, miR‐379 and miR‐432, which associated with hereditary, skeletal, muscular, and developmental disorders.Conclusion:We demonstrate that the repertoire of small RNA molecules in the plasma of children with JIA differs significantly from those seen in the plasma of healthy children. Furthermore, these RNAs show disease‐state specificity, suggesting that they may be directly involved in therapeutic response. Further characterization of these exosomal miRNAs in JIA may provide greater insight into pathobiology and open new doors for the development of blood‐based biomarkers for JIA.

Gamma-Glutamyltransferase Fractions in Human Plasma and Bile: Characteristic and Biogenesis

Total plasma gamma-glutamyltransferase (GGT) activity is a sensitive, non-specific marker of liver dysfunction. Four GGT fractions (b-, m-, s-, f-GGT) were described in plasma and their differential specificity in the diagnosis of liver diseases was suggested. Nevertheless fractional GGT properties have not been investigated yet. The aim of this study was to characterize the molecular nature of fractional GGT in both human plasma and bile.Plasma was obtained from healthy volunteers; whereas bile was collected from patients undergoing liver transplantation. Molecular weight (MW), density, distribution by centrifugal sedimentation and sensitivity to both detergent (deoxycholic acid) and protease (papain) were evaluated. A partial purification of b-GGT was obtained by ultracentrifugation.Plasma b-GGT fraction showed a MW of 2000 kDa and a density between 1.063–1.210 g/ml. Detergent converted b-GGT into s-GGT, whereas papain alone did not produce any effect. Plasma m-GGT and s-GGT showed a MW of 1,000 and 200 kDa, and densities between 1.006-1.063 g/ml and 1.063–1.210 g/ml respectively. Both fractions were unaffected by deoxycholic acid, while GGT activity was recovered into f-GGT peak after papain treatment. Plasma f-GGT showed a MW of 70 kDa and a density higher than 1.21 g/ml. We identified only two chromatographic peaks, in bile, showing similar characteristics as plasma b- and f-GGT fractions.These evidences, together with centrifugal sedimentation properties and immunogold electronic microscopy data, indicate that b-GGT is constituted of membrane microvesicles in both bile and plasma, m-GGT and s-GGT might be constituted of bile-acid micelles, while f-GGT represents the free-soluble form of the enzyme.

Liquid crystal droplet-based amplification of microvesicles that are shed by mammalian cells.

Membrane-derived microvesicles (MVs) shed by cells are being investigated for their role in intercellular communication and as potential biomarkers of disease, but facile and sensitive methods for their analysis do not exist. Here we demonstrate new principles for analysis of MVs that use micrometer-sized droplets of liquid crystals (LCs) to amplify MVs that are selectively captured via antibody-mediated interactions. The influence of the MVs on the micrometer-sized LC droplets is shown to be readily quantified via use of flow cytometry. The methodology was developed using MVs shed by epidermoid carcinoma A431 cells that contain epidermal growth factor receptor (EGFR) as an important and representative example of MVs containing signaling proteins that play a central role in cancer. The LC droplets were found to be sensitive to 10(6) MVs containing EGFR (relative to controls using isotype control antibody) and to possess a dynamic range of response across several orders of magnitude. Because the 100 nm-sized MVs captured via EGFR generate an optical response in the micrometer-sized LC droplets that can be readily detected by flow cytometry in light scattering mode, the approach possesses significant advantages over direct detection of MVs by flow cytometry. The LC droplets are also substantially more sensitive than techniques such as immunoblotting because the lipid-component of the MVs serves to amplify the antibody-mediated capture of the target proteins in the MVs. Other merits of the approach are defined and discussed in the paper.

Experimental Investigations of Single Liposome to Supported Bilayer Binding Events

One of the primary ways in which cells interact with their environments is by release of extracellular vesicles. These cell-derived submicron sized containers are formed either from the cell plasma membrane (microvesicles) or secreted from multivesicular bodies (exosomes). These vesicles contain nucleic acids and proteins that have been suggested to play an important role in intercellular signaling and the process of molecular communication between cells. In microvesicle-mediated intercellular communication, vesicles released by a donor cell must bind to the plasma membrane of a recipient cell in order to deliver their cargo to the target. Despite the important physiological role of vesicle-plasma membrane fusion and vesicle endocytosis, the details of the physical interactions between microvesicles and the plasma membrane are still poorly understood. To better understand the forces which occur between microvesicles and cells, we experimentally investigate single liposome binding events with a supported lipid bilayer. We employed total internal reflection fluorescence (TIRF) microscopy to observe liposome interactions with a supported lipid bilayer, and compare the experimentally observed bond lifetimes to the theoretical estimates. Our experimental observations indicate that bond lifetimes increase with both increasing liposome size and decreasing temperature.

Abstract 18508: Exposure of Adipocytes to High Mobility Group Box 1 Impairs Insulin Signaling via RAGE, While Stimulating Monocyte Recruitment Through TLR2

Background: Smokers show increased prevalence of insulin resistance for glucose and type 2 diabetes mellitus (T2DM). The underlying mechanisms, however, remain poorly understood. Tobacco smoke exposure increases tissue expression of high mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) that activates innate immunity. We recently reported that exposure of human macrophages to tobacco smoke extract (TSE) provokes the release of HMGB1 in two forms: as a soluble molecule and carried on membrane microvesicles (MV). Both soluble recombinant HMGB1 (rHMGB1) and MV-associated HMGB1 on TSE-induced MVs (TSE-MVs) mediate crucial crosstalk with adipocytes by impairing insulin signaling and by recruiting monocytes to the adipocytes - GOAL- To identify the receptors on adipocytes that mediate the effects of HMGB1 on insulin signaling and monocyte recruitment. Methods: Cultured 3T3-L1 adipocytes were incubated for 24h with rHMGB1 or TSE-MVs, in the absence or presence of neutralizing antibodies against different receptors, followed by assays of signaling responses to insulin and chemoattraction of monocytes across a Boyden chamber. Results: Exposure of adipocytes to rHMGB1 or TSE-MVs impaired insulin-induced phosphorylations of IRS1 (Tyr612) and AKT (Ser473) and induced MCP1 secretion and hence monocyte transmigration and adherence to the cultured adipocytes. Neutralizing antibodies against RAGE, but not TLR2, improved insulin signaling in adipocytes exposed to rHMGB1 or TSE-MVs. In contrast, neutralizing antibodies against TLR2, but not RAGE, attenuated the induction of MCP1 secretion, and hence inhibited monocyte transmigration and adherence to cultured adipocytes exposed to rHMGB1 or TSE-MV. Conclusion: Our results indicate that soluble and MV-associated HMGB1 that is released from macrophages exposed to tobacco smoke mediates harmful macrophage-adipocyte crosstalk through two pathways: via adipocyte RAGE to impair insulin signaling, and via adipocyte TLR2 to induce MCP1 secretion and monocyte recruitment. These pathways may contribute to insulin resistance for glucose and the development of T2DM in individuals exposed to tobacco smoke.

Abstract 18994: Shear-Dependent Membrane Scrambling and Microvesiculation and an Important Role for Rac1

Surface exposure of phosphatidylserine (PS) and the release of membrane microvesicles (microparticles) are critically important for blood platelets and leukocytes to promote coagulation and inflammation during vascular injury. The caspase-dependent apoptotic signaling pathway and high intracellular calcium-dependent mechanisms have been implicated in the induction of these events under static conditions. However, it is known that when stimulated with physiological agonists under static conditions only a small percentage of platelets actually respond in a procoagulant manner. Thus, these mechanisms do not fully explain the potent procoagulant response of platelets to physiological stimuli. Here we demonstrate a novel shear stress-dependent mechanism of PS exposure and microvesiculation in platelets stimulated by physiological agonists. We show that exposing agonist-stimulated mouse and human platelets to increasing levels of shear led to greatly increased levels of surfaced exposed PS, shedding of membrane microvesicles, and in particular an increased sensitivity of platelets to display procoagulant activity at low concentrations of physiological agonists. Thus, shear stress is critical for inducing the surface exposure of PS and the shedding of PS-exposed microvesicles in platelets. Moreover, using the Rac1 inhibitor NSC23766 and platelet-specific Rac1 -/- platelets we identified Rac1, a member of the Rho family small GTPases, as being a critical mediator of the platelet procoagulant response to shear. We also show that Rac1 regulates this shear-induced response independent of the known apoptosis signaling pathway, as Rac1 -/- platelets showed no difference in the caspase-dependent pathway of PS exposure induced by ABT737. Importantly, the role of Rac1 in mediating shear-dependent PS exposure and microvesicle release appears to be independent of its known role in stimulating platelet secretion, and represents a common pathway of PS exposure and microvesicle release induced via several different agonists. This novel PS exposure and microvesculation mechanism is important for the function of platelets to promote coagulation.

ARF1 regulates the Rho/MLC pathway to control EGF-dependent breast cancer cell invasion

Invasion of tumor cells is a key step in metastasis that depends largely on the ability of these cells to degrade the extracellular matrix. Although we have showed that the GTPase ADP-ribosylation factor 1 (ARF1) is overexpressed in highly invasive breast cancer cell lines and that epidermal growth factor stimulation can activate this ARF isoform to regulate migration as well as proliferation, the role of this small GTP-binding protein has not been addressed in the context of invasiveness. Here we report that modulation of ARF1 expression and activity markedly impaired the ability of M.D. Anderson-metastatic breast-231 cells, a prototypical highly invasive breast cancer cell line, to degrade the extracellular matrix by controlling metalloproteinase-9 activity. In addition, we demonstrate that this occurs through inhibition of invadopodia maturation and shedding of membrane-derived microvesicles, the two key structures involved in invasion. To further define the molecular mechanisms by which ARF1 controls invasiveness, we show that ARF1 acts to modulate RhoA and RhoC activity, which in turn affects myosin light-chain (MLC) phosphorylation. Together our findings underscore for the first time a key role for ARF1 in invasion of breast cancer cells and suggest that targeting the ARF/Rho/MLC signaling axis might be a promising strategy to inhibit invasiveness and metastasis.

Induction and transport of Wnt 5a during macrophage-induced malignant invasion is mediated by two types of extracellular vesicles

Recently, we have shown that macrophage (MΦ)-induced invasion of breast cancer cells requires upregulation of Wnt 5a in MΦ leading to activation of β-Catenin-independent Wnt signaling in the tumor cells. However, it remained unclear, how malignant cells induce Wnt 5a in MΦ and how it is transferred back to the cancer cells. Here we identify two types of extracellular particles as essential for this intercellular interaction in both directions. Plasma membrane-derived microvesicles (MV) as well as exosomes from breast cancer cells, although biologically distinct populations, both induce Wnt 5a in MΦ. In contrast, the particle-free supernatant and vesicles from benign cells, such as platelets, have no such effect. Induction is antagonized by the Wnt inhibitor Dickkopf-1. Subsequently, Wnt 5a is shuttled via responding MΦ-MV and exosomes to the tumor cells enhancing their invasion. Wnt 5a export on both vesicle fractions depends at least partially on the cargo protein Evenness interrupted (Evi). Its knockdown leads to Wnt 5a depletion of both particle populations and reduced vesicle-mediated invasion. In conclusion, MV and exosomes are critical for MΦ-induced invasion of cancer cells since they are responsible for upregulation of MΦ-Wnt 5a as well as for its delivery to the recipient cells via a reciprocal loop. Although of different biogenesis, both populations share common features regarding function and Evi-dependent secretion of non-canonical Wnts.

Cell Infectivity in Relation to Bovine Leukemia Virus gp51 and p24 in Bovine Milk Exosomes

Exosomes are small membranous microvesicles (40–100 nm in diameter) and are extracellularly released from a wide variety of cells. Exosomes contain microRNA, mRNA, and cellular proteins, which are delivered into recipient cells via these exosomes, and play a role in intercellular communication. In bovine leukemia virus (BLV) infection of cattle, although it is thought to be a minor route of infection, BLV can be transmitted to calves via milk. Here, we investigated the association between exosomes and BLV in bovine milk. BLV structural proteins, gp51 (Env) and p24 (Gag), were detected in bovine milk exosomes from BLV-infected cattle by Western blot analysis. In cells inoculated with these milk exosomes, BLV DNA was not detected during three serial passages by nested PCR. Purification of exosomes from persistently BLV-infected cells was achieved by immuno-magnetic separation using an antibody against exosomes coupled to magnetic beads. Consistently, BLV gp51 and p24 proteins were detected in purified exosomes. Moreover, reverse transcriptase activity was observed in purified exosomes, meaning that exosomes also contain viral enzyme. However, BLV DNA was not detected in serially passaged cells after inoculation of purified exosomes, indicating that exosomes carrying BLV proteins appeared to be not infectious. These results suggest that BLV proteins are released with milk exosomes and could be transferred into recipient cells of calves via milk exosomes as an alternative route not requiring virus infection. Moreover it is also possible that bovine milk exosomes play a role in clearance of BLV proteins from infected cells.

Annexin A2 Promotes the Migration and Invasion of Human Hepatocellular Carcinoma Cells In Vitro by Regulating the Shedding of CD147-Harboring Microvesicles from Tumor Cells

It has been reported that Annexin A2 (ANXA2) is up-regulated in hepatocellular carcinoma (HCC), but the roles of ANXA2 in the migration and invasion of HCC cells have not been determined. In this study, we found that ANXA2-specific siRNA (si-ANXA2) significantly inhibited the migration and invasion of HCC cells co-cultured with fibroblasts in vitro. In addition, the production of MMP-2 by fibroblasts cultured in supernatant collected from si-ANXA2-transfected HCC cells was notably down-regulated. ANXA2 was also found to be co-localized and co-immunoprecipitated with CD147. Further investigation revealed that the expression of ANXA2 in HCC cells affected the shedding of CD147-harboring membrane microvesicles, acting as a vehicle for CD147 in tumor-stromal interactions and thereby regulating the production of MMP-2 by fibroblasts. Together, these results suggest that ANXA2 enhances the migration and invasion potential of HCC cells in vitro by regulating the trafficking of CD147-harboring membrane microvesicles.

Hyaluronan production enhances shedding of plasma membrane-derived microvesicles

Many cell types secrete plasma membrane-bound microvesicles, suggested to play an important role in tissue morphogenesis, wound healing, and cancer spreading. However, the mechanisms of their formation have remained largely unknown. It was found that the tips of long microvilli induced in cells by overexpression of hyaluronan synthase 3 (HAS3) were detach into the culture medium as microvesicles. Moreover, several cell types with naturally active hyaluronan synthesis released high numbers of plasma membrane-derived vesicles, and inhibition of hyaluronan synthesis reduced their formation. The vesicles contained HAS, and were covered with a thick hyaluronan coat, a part of which was retained even after purification with high-speed centrifugation. HAS3 overexpressing MDCK cells cultured in a 3-D matrix as epithelial cysts released large amounts of HAS- and hyaluronan-positive vesicles from their basal surfaces into the extracellular matrix. As far as we know, hyaluronan synthesis is one of the first molecular mechanisms shown to stimulate the production of microvesicles. The microvesicles have a potential to deliver the hyaluronan synthase machinery and membrane and cytoplasmic materials to other cells, influencing tissue regeneration, inflammation and tumor progression.

Characterization of human plasma-derived exosomal RNAs by deep sequencing.

BackgroundExosomes, endosome-derived membrane microvesicles, contain specific RNA transcripts that are thought to be involved in cell-cell communication. These RNA transcripts have great potential as disease biomarkers. To characterize exosomal RNA profiles systemically, we performed RNA sequencing analysis using three human plasma samples and evaluated the efficacies of small RNA library preparation protocols from three manufacturers. In all we evaluated 14 libraries (7 replicates).ResultsFrom the 14 size-selected sequencing libraries, we obtained a total of 101.8 million raw single-end reads, an average of about 7.27 million reads per library. Sequence analysis showed that there was a diverse collection of the exosomal RNA species among which microRNAs (miRNAs) were the most abundant, making up over 42.32% of all raw reads and 76.20% of all mappable reads. At the current read depth, 593 miRNAs were detectable. The five most common miRNAs (miR-99a-5p, miR-128, miR-124-3p, miR-22-3p, and miR-99b-5p) collectively accounted for 48.99% of all mappable miRNA sequences. MiRNA target gene enrichment analysis suggested that the highly abundant miRNAs may play an important role in biological functions such as protein phosphorylation, RNA splicing, chromosomal abnormality, and angiogenesis. From the unknown RNA sequences, we predicted 185 potential miRNA candidates. Furthermore, we detected significant fractions of other RNA species including ribosomal RNA (9.16% of all mappable counts), long non-coding RNA (3.36%), piwi-interacting RNA (1.31%), transfer RNA (1.24%), small nuclear RNA (0.18%), and small nucleolar RNA (0.01%); fragments of coding sequence (1.36%), 5′ untranslated region (0.21%), and 3′ untranslated region (0.54%) were also present. In addition to the RNA composition of the libraries, we found that the three tested commercial kits generated a sufficient number of DNA fragments for sequencing but each had significant bias toward capturing specific RNAs.ConclusionsThis study demonstrated that a wide variety of RNA species are embedded in the circulating vesicles. To our knowledge, this is the first report that applied deep sequencing to discover and characterize profiles of plasma-derived exosomal RNAs. Further characterization of these extracellular RNAs in diverse human populations will provide reference profiles and open new doors for the development of blood-based biomarkers for human diseases.

23. The challenges and limitations of current clinical methods for the hypothermic storage and cryopreservation of human red blood cells

One of the most important advances that led to the creation of blood banks and the widespread therapeutic use of blood products was the development of techniques for ex vivo blood preservation. All efforts to extend the storage of blood past a few days had failed until the discovery of the protective effects of glucose on citrated blood in 1916. Since then, the hypothermic storage of viable red blood cells (RBC) at 4 °C has advanced to the point where these cells can be preserved ex vivo for up to 45 days with modern additive and extender solutions. Cryopreservation of RBCs for transfusion is one of the most successful applications of the cryobiology research that followed the discovery of glycerol as a cryoprotectant. This presentation will review the challenges and limitations of the current methods used in blood banks for the hypothermic storage and cryopreservation of human RBC used in transfusion medicine. Emerging data on the RBC storage lesion and RBC cryoinjury will be discussed with a focus on changes to cell metabolism, oxidative injury and membrane microvesiculation. Current RBC quality control methods and international regulations will be briefly described and details on the Canadian Blood Services RBC Quality Monitoring program will be presented. Recent clinical data on the effects of hypothermic red blood cell storage and cryopreservation on recipient outcomes will be reviewed. While RBC transfusions have been used in the treatment of the anemic patient for almost 100 years, clinical data indicates that there is a need for additional improvements. Applying emerging principles and techniques in the preservation sciences would have important clinical impact on the quality and efficacy of hypothermic stored and cryopreserved RBC.