Shedding Of Microvesicles Research Articles

The Protease Activated Receptor2 Promotes Rab5a Mediated Generation of Pro-metastatic Microvesicles

Metastasis, the hallmark of cancer propagation is attributed by the modification of phenotypic/functional behavior of cells to break attachment and migrate to distant body parts. Cancer cell-secreted microvesicles (MVs) contribute immensely in disease propagation. These nano-vesicles, generated from plasma membrane outward budding are taken up by nearby healthy cells thereby inducing phenotypic alterations in those recipient cells. Protease activated receptor 2 (PAR2), activated by trypsin, also contributes to cancer progression by increasing metastasis, angiogenesis etc. Here, we report that PAR2 activation promotes pro-metastatic MVs generation from human breast cancer cell line, MDA-MB-231. Rab5a, located at the plasma membrane plays vital roles in MVs biogenesis. We show that PAR2 stimulation promotes AKT phosphorylation which activates Rab5a by converting inactive Rab5a-GDP to active Rab5a-GTP. Active Rab5a polymerizes actin which critically regulates MVs shedding. Not only MVs generation, has this Rab5a activation also promoted cell migration and invasion. We reveal that Rab5a is over-expressed in human breast tumor specimen and contributes MVs generation in those patients. The involvement of p38 MAPK in MVs-induced cell metastasis has also been highlighted in the present study. Blockade of Rab5a activation can be a potential therapeutic approach to restrict MVs shedding and associated breast cancer metastasis.

Enhanced Th1 and inflammatory mRNA responses upregulate NK cell cytotoxicity and NKG2D ligand expression in human pre-eclamptic placenta and target it for NK cell attack.

Pre-eclampsia (PE), a severe human pregnancy disorder, is associated with exaggerated systemic inflammation, enhanced cytokine production, and increased shedding of microvesicles leading to endothelial dysfunction, coagulopathy, and extensive placenta destruction. The cause of PE is still unclear. Evidence suggests that its origin lies in the placenta and that the maternal immune system is involved. A shift in cytokine production in PE pregnancy promotes NK cell activation, suggested to be important in PE pathogenesis. In line with this suggestion, we studied NK cell cytotoxicity in peripheral blood of PE patients and controls and the mRNA expression of cytokines and of the NKG2D receptor and its ligands MICA/B and ULBP1-3 in PE- and normal placenta. The cytotoxic capacity of peripheral blood NK cells was analyzed using K562 target cells. The cytokine mRNA profiles and the mRNA expression of the NKG2D receptor and its ligands MICA/B and ULBP 1-3 in PE placenta were assessed and compared to those in normal placenta using real-time quantitative RT-PCR. The cytotoxicity of peripheral blood NK cells was upregulated in PE cases. Further, we found an enhanced inflammatory cytokine mRNA response combined with a dysregulated regulatory response and a significant mRNA overexpression of NKG2D receptor and its ligands MICA/B and ULBP in PE placenta. The destruction of chorionic villi observed in PE placenta might be conveyed by an enhanced local cytotoxic response through the NKG2D receptor-ligand pathway, which in turn might be promoted by an intense inflammatory response not counteracted by regulatory cytokine responses.

Functional Association between Regulatory RNAs and the Annexins.

Cells respond to pathophysiological states by activation of stress-induced signalling. Regulatory non-coding microRNAs (miRNAs) often form stable feed-forward loops which ensure prolongation of the signal, contributing to sustained activation. Members of the annexin protein family act as sensors for Ca2+, pH, and lipid second messengers, and regulate various signalling pathways. Recently, annexins were reported to participate in feedback loops, suppressing miRNA synthesis and attenuating stress-induced dysregulation of gene expression. They can directly or indirectly associate with RNAs, and are transferred between the cells in exosomes and shed microvesicles. The ability of annexins to recruit other proteins and miRNAs into exosomes implicates them in control of cell–cell interactions, affecting the adaptive responses and remodelling processes during disease. The studies summarized in this Review point to an emerging role of annexins in influencing the synthesis, localisation, and transfer of regulatory RNAs.

Mechanisms and modulation of microvesicle uptake in a model of alveolar cell communication

Extracellular vesicles, including exosomes and shed microvesicles (MVs), can be internalized by recipient cells to modulate function. Although the mechanism by which extracellular vesicles are internalized is incompletely characterized, it is generally considered to involve endocytosis and an initial surface-binding event. Furthermore, modulation of uptake by microenvironmental factors is largely unstudied. Here, we used flow cytometry, confocal microscopy, and pharmacologic and molecular targeting to address these gaps in knowledge in a model of pulmonary alveolar cell-cell communication. Alveolar macrophage-derived MVs were fully internalized by alveolar epithelial cells in a time-, dose-, and temperature-dependent manner. Uptake was dependent on dynamin and actin polymerization. However, it was neither saturable nor dependent on clathrin or receptor binding. Internalization was enhanced by extracellular proteins but was inhibited by cigarette smoke extract via oxidative disruption of actin polymerization. We conclude that MV internalization occurs via a pathway more consistent with fluid-phase than receptor-dependent endocytosis and is subject to bidirectional modulation by relevant pathologic perturbations.

Shedding of Microvesicles from Microglia Contributes to the Effects Induced by Metabotropic Glutamate Receptor 5 Activation on Neuronal Death.

Metabotropic glutamate (mGlu) receptor 5 is involved in neuroinflammation and has been shown to mediate reduced inflammation and neurotoxicity and to modify microglia polarization. On the other hand, blockade of mGlu5 receptor results in inhibition of microglia activation. To dissect this controversy, we investigated whether microvesicles (MVs) released from microglia BV2 cells could contribute to the communication between microglia and neurons and whether this interaction was modulated by mGlu5 receptor. Activation of purinergic ionotropic P2X7 receptor with the stable ATP analog benzoyl-ATP (100 μM) caused rapid MVs shedding from BV2 cells. Ionic currents through P2X7 receptor increased in BV2 cells pretreated for 24 h with the mGlu5 receptor agonist CHPG (200 μM) as by patch-clamp recording. This increase was blunted when microglia cells were activated by exposure to lipopolysaccharide (LPS; 0.1 μg/ml for 6 h). Accordingly, a greater amount of MVs formed after CHPG treatment, an effect prevented by the mGlu5 receptor antagonist MTEP (100 μM), as measured by expression of flotillin, a membrane protein enriched in MVs. Transferred MVs were internalized by SH-SY5Y neurons where they did not modify neuronal death induced by a low concentration of rotenone (0.1 μM for 24 h), but significantly increased rotenone neurotoxicity when shed from CHPG-treated BV2 cells. miR146a was increased in CHPG-treated MVs, an effect concealed in MVs from LPS-activated BV2 cells that showed per se an increase in miRNA146a levels. The present data support a role for microglia-shed MVs in mGlu5-mediated modulation of neuronal death and identify miRNAs as potential critical mediators of this interaction.

Placental hypoxia inducible factor -1α & CHOP immuno-histochemical expression relative to maternal circulatory syncytiotrophoblast micro-vesicles in preeclamptic and normotensive pregnancies

ObjectivesPreeclampsia (PE) occurs as a result placental hypoxia-induced oxidative and endoplasmic reticulum stress, and is associated with the activation of hypoxia inducible factor-1α (HIF-1α) and apoptotic CHOP pathways with the consequential shedding of syncytiotrophoblast microvesicles which may be central in mediating the maternal systemic immune response. The aim of this study was to immune-localise and morphometrically analyse CHOP and HIF-1α within the placenta of normotensive and pre-eclamptic pregnancies and concomitantly quantify syncytiotrophoblast released microvesicles in maternal circulation. Study designPlacental tissue and plasma were obtained from normotensive and pre-eclamptic pregnant women. The expression of CHOP and HIF-1α was analysed using immunohistochemistry. Isolation and size distribution of the circulating maternal microvesicles was determined using nanoparticle tracking analysis. The concentration of syncytiotrophoblast microvesicles was determined using the placental alkaline phosphatase ELISA. ResultsThis study demonstrates a significant increase in immunohistochemical expression of HIF-1 α and CHOP in preeclampsia compared to the normotensive women (p<0.05). In keeping with this, a significant increase in the mean syncytiotrophoblast microvesicles concentration was observed in PE, compared to normotensives (p<0.05). A positive correlation between placental expression of CHOP and HIF-1α and STBMs was obtained. ConclusionThis study demonstrates increased placental expression of HIF-1α and CHOP in preeclampsia compared to normotensive pregnancies which correlate to their increased syncytiotrophoblast microvesicles concentration in maternal circulation. These findings indicate that placental hypoxia and ER stress are interrelated contributory factors to the pathogenesis of PE and the consequential release of placental derived debris into the maternal circulation.

Bone Marrow Dendritic Cells Derived Microvesicles for Combinational Immunochemotherapy against Tumor

AbstractVarious types of cell can change the cytoskeleton and shed microvesicles (MVs) with biomimic properties as parent cells in response to stimuli. To take use of the drug package capability of MVs and the potent antigen presentation property of dendritic cells (DCs), DC‐derived antigenic MVs are constructed by priming DCs with tumor‐derived MVs and then encapsulating a chemotherapeutic drug during MVs shedding. This kind of MVs exhibit significant inhibition on melanoma tumor growth and metastasis. The MV‐encapsulated chemotherapeutics can induce direct cytotoxicity and immunogenic cell death in tumor cells. Moreover, a robust antitumor immunity is induced in both, the tumor‐draining lymph node and the tumor microenvironment as the infiltration and activation of T lymphocytes increases. This kind of MVs is further explored in a hepatic ascites model with remarkable prolonged overall survival of mice. More importantly, the MVs can extend the survival of 60% mice more than 150 d without ascites even after rechallenging the tumor twice. This study demonstrates that antigenic MVs with chemotherapeutics possess great potential in cancer immunochemotherapy.

Sphingomyelin encrypts tissue factor: ATP-induced activation of A-SMase leads to tissue factor decryption and microvesicle shedding.

A majority of tissue factor (TF) on cell surfaces exists in an encrypted state with minimal to no procoagulant activity. At present, it is unclear whether limited availability of phosphatidylserine (PS) and/or a specific membrane lipid in the outer leaflet of the plasma membrane contributes to TF encryption. Sphingomyelin (SM) is a major phospholipid in the outer leaflet, and SM metabolism is shown to be altered in many disease settings that cause thrombotic disorders. The present study is carried out to investigate the effect of SM metabolism on TF activity and TF+ microvesicles (MVs) release. In vitro studies using TF reconstituted into liposomes containing varying molar ratios of SM showed that a high molar ratio of SM in the proteoliposomes inhibits TF coagulant activity. Treatment of macrophages with sphingomyelinase (SMase) that hydrolyzes SM in the outer leaflet results in increased TF activity at the cell surface and TF+ MVs release without increasing PS externalization. Adenosine triphosphate (ATP) stimulation of macrophages that activates TF and induces MV shedding also leads to translocation of acid-sphingomyelinase (A-SMase) to the plasma membrane. ATP stimulation increases the hydrolysis of SM in the outer leaflet. Inhibition of A-SMase expression or activity not only attenuates ATP-induced SM hydrolysis, but also inhibits ATP-induced TF decryption and TF+ MVs release. Overall, our novel findings show that SM plays a role in maintaining TF in an encrypted state in resting cells and hydrolysis of SM following cell injury removes the inhibitory effect of SM on TF activity, thus leading to TF decryption.

Psychosine enhances the shedding of membrane microvesicles: Implications in demyelination in Krabbe's disease.

In prior studies, our laboratory showed that psychosine accumulates and disrupts lipid rafts in brain membranes of Krabbe’s disease. A model of lipid raft disruption helped explaining psychosine’s effects on several signaling pathways important for oligodendrocyte survival and differentiation but provided more limited insight in how this sphingolipid caused demyelination. Here, we have studied how this cationic inverted coned lipid affects the fluidity, stability and structure of myelin and plasma membranes. Using a combination of cutting-edge imaging techniques in non-myelinating (red blood cell), and myelinating (oligodendrocyte) cell models, we show that psychosine is sufficient to disrupt sphingomyelin-enriched domains, increases the rigidity of localized areas in the plasma membrane, and promotes the shedding of membranous microvesicles. The same physicochemical and structural changes were measured in myelin membranes purified from the mutant mouse Twitcher, a model for Krabbe’s disease. Areas of higher rigidity were measured in Twitcher myelin and correlated with higher levels of psychosine and of myelin microvesiculation. These results expand our previous analyses and support, for the first time a pathogenic mechanism where psychosine’s toxicity in Krabbe disease involves deregulation of cell signaling not only by disruption of membrane rafts, but also by direct local destabilization and fragmentation of the membrane through microvesiculation. This model of membrane disruption may be fundamental to introduce focal weak points in the myelin sheath, and consequent diffuse demyelination in this leukodystrophy, with possible commonality to other demyelinating disorders.

TRPV1 channels are critical brain inflammation detectors and neuropathic pain biomarkers in mice

The capsaicin receptor TRPV1 has been widely characterized in the sensory system as a key component of pain and inflammation. A large amount of evidence shows that TRPV1 is also functional in the brain although its role is still debated. Here we report that TRPV1 is highly expressed in microglial cells rather than neurons of the anterior cingulate cortex and other brain areas. We found that stimulation of microglial TRPV1 controls cortical microglia activation per se and indirectly enhances glutamatergic transmission in neurons by promoting extracellular microglial microvesicles shedding. Conversely, in the cortex of mice suffering from neuropathic pain, TRPV1 is also present in neurons affecting their intrinsic electrical properties and synaptic strength. Altogether, these findings identify brain TRPV1 as potential detector of harmful stimuli and a key player of microglia to neuron communication.

Emmprin, released as a microvesicle in epithelioid sarcoma, interacts with fibroblasts.

Emmprin (extracellular matrix metalloproteinase inducer, CD147) is a glycosylated transmembrane protein, consisting of two immunoglobulin domains, that stimulates the production of matrix metalloproteinases (MMPs) by tumor-associated fibroblasts. These effects play important roles in tumor invasion and metastasis. However, the precise mechanisms by which emmprin acts on fibroblasts have not been fully elucidated, especially in sarcoma cells. Previously, we demonstrated that emmprin, expressed in conditioned medium collected from the epithelioid sarcoma cell line (FU-EPS-1), stimulates MMP-2 production via interactions with fibroblasts. In this study, we used microvesicles derived from sarcoma cells, and determined whether emmprin exists in the microvesicles, which enhance the production of MMP-2 via fibroblasts. Microvesicles released from FU-EPS-1 cells were shown to contain full-length emmprin, identified as a 45-kDa protein characterized by polylactosamine glycosylation. Microvesicles collected from FU-EPS-1 cells transfected with emmprin-specific siRNA or transduced with shRNA displayed significantly reduced MMP-2 production by fibroblasts compared with those from control-transfected cells. Our findings show that emmprin is released through microvesicle shedding in sarcoma cells, and emmprin in microvesicles regulates MMP-2 production by influencing the activity of fibroblasts located at sites distant from the tumor cells.

LB06 - Mesenchymal stem cells shuttle micrornas via extracellular vesicles and prime resistant GBM to caspase mediated apoptosis

Mesenchymal stem cell (MSC)-based therapies have yielded beneficial effects in a broad range of animal models of disease and human clinical trials. MSC shed extracellular vesicles and microvesicles into the extracellular space that contain small non-coding RNA molecules, microRNAs (miR). In this study, we engineered MSC to shed exosomes containing microRNA that control cell proliferation and apoptosis in tumors and investigated their potential to prime resistant tumor cells to apoptosis in glioblastoma (GBM) brain tumors. We show that exosomes isolated from engineered MSC are enriched in miR-1 and miR-7and are efficiently transferred into the GMB stem cells (GSC) via exosomes. In order to develop a therapeutic strategy utilizing miRs,,we created constitutive and regulatable miR-7 expression vectors and utilized pharmacological inhibition of caspases and genetic loss of function to study the effect of forced expression of miR-7 on death receptor (DR) pathways in a cohort of death receptor ligand (DRL) resistant established GBM and patient-derived primary GSC. We show that expression of miR-7 results in the up-regulation of death receptor (DR) 5 and primes DRL resistant GBM cells to DRL, tumor necrosis factor apoptosis inducing ligand (TRAIL) mediated apoptotic cell death. In an effort to explore the mechanism underlying the cross talk between miR-7 targeted cell proliferation pathway and TRAIL/death receptor (DR) 4/5 mediated extrinsic apoptotic pathway; we show the interaction between various downstream players involving the NF-kB signaling and their role in up-regulating death receptors thereby priming them to DRLmediated apoptotic cell death. In vivo, we show that miR-7 significantly decreases tumor volumes and when combined with MSC-S-TRAIL, impairs tumor progression and significantly prolongs of mice bearing patient-derived GBM. In conclusion, our findings shed light on the presence anti-tumorgenic miRs in MSC shed exosomes and a unique role of miR-7 in priming resistant GBM cells to DRL mediated apoptosis. This study serves as a foundation for developing therapies utilizing engineered MSC to deliver a combination of microRNA and receptor targeted ligands for cancer therapy.

Mesenchymal Stem Cell-Derived Microvesicles Modulate Lipopolysaccharides-Induced Inflammatory Responses to Microglia Cells.

Microglia cells are the central nervous system immune cells and have been pointed out as the main mediators of the inflammation leading to neurodegenerative disorders. Mesenchymal stromal cells (MSCs) are a heterogeneous population of cells with very high self-renewal properties and uncomplicated in vitro culture. Research has shown that MSCs have the capacity to induce tissue regeneration and reduce inflammation. Studies demonstrated that MSCs have complex paracrine machineries involving shedding of cell-derived microvesicles (MVs), which entail part of the regulatory and regenerative activity of MSCs, as observed in animal models. We proposed MSC-derived MVs as potent regulators of microglia activation and used an in vitro model of stimulation for BV-2 cells, a microglia cell line, with lipopolysaccharides (LPS). Here we demonstrated that presence of MSCs-derived MVs (MSC-MVs) prevents Tumor necrosis factor-α, Interleukin (IL)-1β and IL-6 upregulation by BV-2 cells and primary microglia cells toward LPS. Also, inducible isoform of nitric oxide synthases and Prostaglandin-endoperoxide synthase 2 upregulation were hampered in presence of MSC-MVs. Higher levels of the M2 microglia marker chemokine ligand-22 were detectable in BV-2 cells after coculture with MSC-MVs in presence and absence of LPS. Moreover, upregulation of the activation markers CD45 and CD11b by BV-2 cells was prevented when cocultured with MSC-MVs. Furthermore, MSC-MVs suppressed the phosphorylation of the extracellular signal kinases 1/2, c-Jun N-terminal kinases and the p38 MAP kinase (p38) molecules. Consequently, MSC-MVs might represent a modulator of microglia activation with future therapeutic impact. Stem Cells 2017;35:812-823.

Intrinsic repair protects cells from pore-forming toxins by microvesicle shedding.

Pore-forming toxins (PFTs) are used by both the immune system and by pathogens to disrupt cell membranes. Cells attempt to repair this disruption in various ways, but the exact mechanism(s) that cells use are not fully understood, nor agreed upon. Current models for membrane repair include (1) patch formation (e.g., fusion of internal vesicles with plasma membrane defects), (2) endocytosis of the pores, and (3) shedding of the pores by blebbing from the cell membrane. In this study, we sought to determine the specific mechanism(s) that cells use to resist three different cholesterol-dependent PFTs: Streptolysin O, Perfringolysin O, and Intermedilysin. We found that all three toxins were shed from cells by blebbing from the cell membrane on extracellular microvesicles (MVs). Unique among the cells studied, we found that macrophages were 10 times more resistant to the toxins, yet they shed significantly smaller vesicles than the other cells. To examine the mechanism of shedding, we tested whether toxins with engineered defects in pore formation or oligomerization were shed. We found that oligomerization was necessary and sufficient for membrane shedding, suggesting that calcium influx and patch formation were not required for shedding. However, pore formation enhanced shedding, suggesting that calcium influx and patch formation enhance repair. In contrast, monomeric toxins were endocytosed. These data indicate that cells use two interrelated mechanisms of membrane repair: lipid-dependent MV shedding, which we term 'intrinsic repair', and patch formation by intracellular organelles. Endocytosis may act after membrane repair is complete by removing inactivated and monomeric toxins from the cell surface.

Extracellular vesicles: their role in cancer biology and epithelial-mesenchymal transition.

Cell-cell communication is critical across an assortment of physiological and pathological processes. Extracellular vesicles (EVs) represent an integral facet of intercellular communication largely through the transfer of functional cargo such as proteins, messenger RNAs (mRNAs), microRNA (miRNAs), DNAs and lipids. EVs, especially exosomes and shed microvesicles, represent an important delivery medium in the tumour micro-environment through the reciprocal dissemination of signals between cancer and resident stromal cells to facilitate tumorigenesis and metastasis. An important step of the metastatic cascade is the reprogramming of cancer cells from an epithelial to mesenchymal phenotype (epithelial-mesenchymal transition, EMT), which is associated with increased aggressiveness, invasiveness and metastatic potential. There is now increasing evidence demonstrating that EVs released by cells undergoing EMT are reprogrammed (protein and RNA content) during this process. This review summarises current knowledge of EV-mediated functional transfer of proteins and RNA species (mRNA, miRNA, long non-coding RNA) between cells in cancer biology and the EMT process. An in-depth understanding of EVs associated with EMT, with emphasis on molecular composition (proteins and RNA species), will provide fundamental insights into cancer biology.

A Protocol for Isolation and Proteomic Characterization of Distinct Extracellular Vesicle Subtypes by Sequential Centrifugal Ultrafiltration.

Scientific and clinical interest in extracellular vesicles (EVs) has increased rapidly as evidence mounts that they may constitute a new signaling paradigm. Recent studies have highlighted EVs carry preassembled complex biological information that elicit pleiotropic responses in target cells. It is well recognized that cells secrete essentially two EV subtypes that can be partially separated by differential centrifugation (DC): the larger size class (referred to as "microvesicles" or "shed microvesicles," sMVs) is heterogeneous (100-1500 nm), while the smaller size class (referred to as "exosomes") is relatively homogeneous in size (50-150 nm). A key issue hindering progress in understanding underlying mechanisms of EV subtype biogenesis and cargo selectivity has been the technical challenge of isolating homogeneous EV subpopulations suitable for molecular analysis. In this protocol we reveal a novel method for the isolation, purification, and characterization of distinct EV subtypes: exosomes and sMVs. This method, based on sequential centrifugal ultrafiltration (SCUF), affords unbiased isolation of EVs from conditioned medium from a human colon cancer cell model. For both EV subtypes, this protocol details extensive purification and characterization based on dynamic light scattering, cryoelectron microscopy, quantitation, immunoblotting, and comparative label-free proteome profiling. This analytical SCUF method developed is potentially scalable using tangential flow filtration and provides a solid foundation for future in-depth functional studies of EV subtypes from diverse cell types.

Transcriptome and long noncoding RNA sequencing of three extracellular vesicle subtypes released from the human colon cancer LIM1863 cell line.

Previously we reported that LIM1863 colorectal cancer (CRC) cells secrete three distinct extracellular vesicle subtypes – two subpopulations of exosomes (apical EpCAM-Exos and basolateral A33-Exos) and shed microvesicles (sMVs) – with distinct protein and miRNA signatures. Here, we extend our omics approach to understand the fundamental role of LIM1863-derived EVs by performing a comprehensive analysis of their mRNAs and long non-coding RNAs (lncRNAs) using RNA-Seq. We show that 2,389 mRNAs, 317 pseudogene transcripts, 1,028 lncRNAs and 206 short non-coding RNAs selectively distributed to (i.e., are enriched in) LIM1863 EVs, relative to the parent cell. An Ensembl/UniProtKB analysis revealed 1,937 mRNAs encode canonical proteins, 348 isoforms (including splice-variant proteins), and 119 ‘missing proteins’ (i.e., annotated in Ensembl but not UniProtKB). Further dissection of our protein/RNA data revealed that 6/151 observed RNA binding proteins have the potential to interact with ~75% of EV-enriched RNAs. Intriguingly, the co-existence of U1 and U2 ribonucleoproteins and their cognate snRNAs in LIM1863 EVs suggests a possible association of CRC EVs with recipient cell splicing events. Our data reveal several potential lncRNA CRC biomarkers and novel splicing/fusion genes that, collectively, will advance our understanding of EV biology in CRC and accelerate the development of EV-based diagnostics and therapeutics.

Microvesicles shed from microglia activated by the P2X7-p38 pathway are involved in neuropathic pain induced by spinal nerve ligation in rats

Microglia are critical in the pathogenesis of neuropathic pain. In this study, we investigated the role of microvesicles (MVs) in neuropathic pain induced by spinal nerve ligation (SNL) in rats. First, we found that MVs shed from microglia were increased in the cerebrospinal fluid and dorsal horn of the spinal cord after SNL. Next, MVs significantly reduced paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). In addition, the P2X7-p38 pathway was related to the bleb of MVs after SNL. Interleukin (IL)-1β was found to be significantly upregulated in the package of MVs, and PWT and PWL increased following inhibition with shRNA-IL-1β. Finally, the amplitude and frequency of spontaneous excitatory postsynaptic currents increased following stimulation with MVs. Our results indicate that the P2X7-p38 pathway is closely correlated with the shedding of MVs from microglia in neuropathic pain, and MVs had a significant effect on neuropathic pain by participating in the interaction between microglia and neurons.

Active release of pneumolysin prepores and pores by mammalian cells undergoing a Streptococcus pneumoniae attack

BackgroundStreptococcus pneumoniae is a potent human pathogen. Its pore-forming exotoxin pneumolysin is instrumental for breaching the host's epithelial barrier and for the incapacitation of the immune system. Methods and resultsUsing a combination of life imaging and cryo-electron microscopy we show that pneumolysin, released by cultured bacteria, is capable of permeabilizing the plasmalemma of host cells. However, such permeabilization does not lead to cell lysis since pneumolysin is actively removed by the host cells. The process of pore elimination starts with the formation of pore-bearing plasmalemmal nanotubes and proceeds by the shedding of pores that are embedded in the membrane of released microvesicles. Pneumolysin prepores are likewise removed. The protein composition of the toxin-induced microvesicles, assessed by mass spectrometry, is suggestive of a Ca2+-triggered mechanism encompassing the proteins of the annexin family and members of the endosomal sorting complex required for transport (ESCRT) complex. ConclusionsS. pneumoniae releases sufficient amounts of pneumolysin to perforate the plasmalemma of host cells, however, the immediate cell lysis, which is frequently reported as a result of treatment with purified and artificially concentrated toxin, appears to be an unlikely event in vivo since the toxin pores are efficiently eliminated by microvesicle shedding. Therefore the dysregulation of cellular homeostasis occurring as a result of transient pore formation/elimination should be held responsible for the damaging toxin action. General significanceWe have achieved a comprehensive view of a general plasma membrane repair mechanism after injury by a major bacterial toxin.

Hypoxia regulates the expression of tissue factor pathway signaling elements in a rat glioma model.

Hypoxia and necrosis are fundamental features of glioma, and their emergence is critical for the rapid biological progression of this fatal tumor. The presence of vaso-occlusive thrombus is higher in grade IV tumors [glioblastoma multiforme (GBM)] compared with lower grade tumors, suggesting that the procoagulant properties of the tumor contribute to its aggressive behavior, as well as the establishment of tumor hypoxia and necrosis. Tissue factor (TF), the primary cellular initiator of coagulation, is overexpressed in GBMs and likely favors a thrombotic microenvironment. Phosphatase and tensin homolog (PTEN) loss and hypoxia are two common alterations observed in glioma that may be responsible for TF upregulation. In the present study, ST1 and P7 rat glioma lines, with different levels of aggressiveness, were comparatively analyzed with the aim of identifying differences in procoagulant mechanisms. The results indicated that P7 cells display potent procoagulant activity compared with ST1 cells. Flow cytometric analysis showed less pronounced levels of TF in ST1 cells compared with P7 cells. Notably, P7 cells supported factor X (FX) activation via factor VIIa, whereas no significant FXa generation was observed in ST1 cells. Furthermore, the exposure of phosphatidylserine on the surface of P7 and ST1 cells was investigated. The results supported the assembly of prothrombinase complexes, accounting for the production of thrombin. Furthermore, reverse transcription-quantitative polymerase chain reaction showed that CoCl2 (known to induce a hypoxic-like stress) led to an upregulation of TF levels in P7 and ST1 cells. Therefore, increased TF expression in P7 cells was accompanied by increased TF procoagulant activity. In addition, hypoxia increased the shedding of procoagulant TF-bearing microvesicles in both cell lines. Finally, hypoxic stress induced by treatment with CoCl2 upregulated the expression of the PAR1 receptor in both P7 and ST1 cells. In addition to PAR1, P7, but not ST1 cells, expressed higher levels of PAR2 under hypoxic stress. Thus, modulating these molecular interactions may provide additional insights for the development of more efficient therapeutic strategies against aggressive glioma.