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Telocytes (TCs), a novel interstitial cell entity promoting tissue regeneration, have been described in various tissues. Their role in inter-cellular signalling and tissue remodelling has been reported in almost all human tissues. This study hypothesizes that TC also contributes to tissue remodelling and regeneration of the human thoracic aorta (HTA). The understanding of tissue homeostasis and regenerative potential of the HTA is of high clinical interest as it plays a crucial role in pathogenesis from aortic dilatation to lethal dissection. Therefore, we obtained twenty-five aortic specimens of heart donors during transplantation. The presence of TCs was detected in different layers of aortic tissue and characterized by immunofluorescence and transmission electron microscopy. Further, we cultivated and isolated TCs in highly differentiated form identified by positive staining for CD34 and c-kit. Aortic-derived TC was characterized by the expression of PDGFR-α, PDGFR-β, CD29/integrin β-1 and αSMA and the stem cell markers Nanog and KLF-4. Moreover, TC exosomes were isolated and characterized for soluble angiogenic factors by Western blot. CD34 + /c-kit + TCs shed exosomes containing the soluble factors VEGF-A, KLF-4 and PDGF-A. In summary, TC occurs in the aortic wall. Correspondingly, exosomes, derived from aortic TCs, contain vasculogenesis-relevant proteins. Understanding the regulation of TC-mediated aortic remodelling may be a crucial step towards designing strategies to promote aortic repair and prevent adverse remodelling., (© 2021 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Helium inhalation induces cardioprotection against ischemia/reperfusion injury, the cellular mechanism of which remains not fully elucidated. Extracellular vesicles (EVs) are cell-derived, nano-sized membrane vesicles which play a role in cardioprotective mechanisms, but their function in helium conditioning (HeC) has not been studied so far. We hypothesized that HeC induces fibroblast-mediated cardioprotection via EVs. We isolated neonatal rat cardiac fibroblasts (NRCFs) and exposed them to glucose deprivation and HeC rendered by four cycles of 95% helium + 5% CO 2 for 1 h, followed by 1 h under normoxic condition. After 40 h of HeC, NRCF activation was analyzed with a Western blot (WB) and migration assay. From the cell supernatant, medium extracellular vesicles (mEVs) were isolated with differential centrifugation and analyzed with WB and nanoparticle tracking analysis. The supernatant from HeC-treated NRCFs was transferred to naïve NRCFs or immortalized human umbilical vein endothelial cells (HUVEC-TERT2), and a migration and angiogenesis assay was performed. We found that HeC accelerated the migration of NRCFs and did not increase the expression of fibroblast activation markers. HeC tended to decrease mEV secretion of NRCFs, but the supernatant of HeC or the control NRCFs did not accelerate the migration of naïve NRCFs or affect the angiogenic potential of HUVEC-TERT2. In conclusion, HeC may contribute to cardioprotection by increasing fibroblast migration but not by releasing protective mEVs or soluble factors from cardiac fibroblasts.

Hantaan viruses (HTNVs) are zoonotic pathogens transmitted mainly by rodents and capable of infecting humans. Increasing knowledge of the human response to HTNV infection can guide the development of new preventative vaccines and therapeutic strategies. Here, we show that HTNV can infect CD8 + T cells in vivo in patients diagnosed with hemorrhagic fever with renal syndrome (HFRS). Electron microscopy-mediated tracking of the life cycle and ultrastructure of HTNV-infected CD8 + T cells in vitro showed an association between notable increases in cytoplasmic multivesicular bodies and virus production. Notably, based on a clinical cohort of 280 patients, we found that circulating HTNV-infected CD8 + T cell numbers in blood were proportional to disease severity. These results demonstrate that viral infected CD8 + T cells may be used as an adjunct marker for monitoring HFRS disease progression and that modulating T cell functions may be explored for new treatment strategies.

Introduction: Microvesicles (MVs) are bioactive, submicron-sized (0.01-1000 nm) membrane vesicles released from various types of cells under normal physiological and pathophysiological conditions. MVs have emerged as important mediators of cell-to-cell communication in a diverse range of normal and pathological processes. MVs have been recognized as potential biomarkers in coagulation, inflammation, and cancer. However, for clinical use, minimizing factors which could affect enumeration and phenotypic characterization of MVs during pre-analytical steps is crucial. In this study, we used flow cytometry and nanoparticle tracking analysis (NTA) to investigate the impact of blood collection using with and without anticoagulant on the number and phenotype of MVs in blood samples., Methods: Blood from 30 healthy volunteers was collected by venipuncture into 3.2% sodium citrate and clot activator tubes. MV subpopulations and their concentrations were investigated using flow cytometry and NTA. MV morphology was examined by transmission electron microscopy., Results: Results showed that the concentration of MVs was significantly lower in serum than in plasma and that CD41 + MV, CD41 + /CD62P + MV, CD45 + MV, and CD142 + MV levels from serum were significantly lower than those from plasma, whereas no significant differences in Annexin V (Anx V) + MV, CD235a + MV, and CD144 + MV levels were found. Interestingly, serum MVs had a higher proportion of small-sized MVs and lower proportion of large-sized MVs than did plasma MVs., Conclusion: Although plasma samples are commonly used, our results suggest that serum can also be used in enumeration of MVs, but care must be taken if coagulation is an aspect of the research., (© 2020 John Wiley & Sons Ltd.)

Robust, well-characterized methods for purifying small extracellular vesicles (sEV) from blood are needed before their potential as disease biomarkers can be realized. Here, we compared isolation of sEV from serum by differential ultracentrifugation (DUC) and by exclusion chromatography using commercially available Exo-spin™ columns. We show that sEV can be purified by both methods but Exo-spin™ columns contain copious additional particles recorded by nanoparticle tracking analysis, invalidating its use for quantifying yields. DUC samples contained higher concentrations of exosome specific proteins CD9, CD63 and CD81 and electron microscopy confirmed that most particles in DUC preparations were sEV, whereas Exo-spin™ samples also contained copious co-purified plasma lipids. MACSPlex bead analysis identified multiple exosome surface proteins, with stronger signals in DUC samples, enabling detection of 21 of 37, compared to only 10 in Exo-spin™ samples. Nevertheless, the pattern of expression was consistent in both preparations, indicating that lipids do not interfere with bead-based technologies. Thus, both DUC and Exo-spin™ can be used to isolate sEV from human serum and what is most appropriate depends on the subsequent use of sEV. In summary, Exo-spin™ enables isolation of sEV from blood with vesicle populations similar to the ones recovered by DUC, but with lower concentrations., Competing Interests: The authors declare no conflict of interest.

Rolling neutrophils form tethers with submicron diameters. Here, we report that these tethers detach, forming elongated neutrophil-derived structures (ENDS) in the vessel lumen. We studied ENDS formation in mice and humans in vitro and in vivo. ENDS do not contain mitochondria, endoplasmic reticulum, or DNA, but are enriched for S100A8, S100A9, and 57 other proteins. Within hours of formation, ENDS round up, and some of them begin to present phosphatidylserine on their surface (detected by annexin-5 binding) and release S100A8-S100A9 complex, a damage-associated molecular pattern protein that is a known biomarker of neutrophilic inflammation. ENDS appear in blood plasma of mice upon induction of septic shock. Compared with healthy donors, ENDS are 10-100-fold elevated in blood plasma of septic patients. Unlike neutrophil-derived extracellular vesicles, most ENDS are negative for the tetraspanins CD9, CD63, and CD81. We conclude that ENDS are a new class of bloodborne submicron particles with a formation mechanism linked to neutrophil rolling on the vessel wall., Competing Interests: Disclosures: The authors declare no competing interests exist., (© 2020 Marki et al.)

Cell-derived Drug Delivery Systems (DDSs), particularly exosomes, have grown in popularity and have been increasingly explored as novel DDSs, due to their intrinsic targeting capabilities. However, clinical translation of exosomes is impeded by the tedious isolation procedures and poor yield. Cell-derived nanovesicles (CDNs) have recently been produced and proposed as exosome-mimetics. Various methods for producing exosome-mimetics have been developed. In this chapter, we present a simple, efficient, and cost-effective CDNs production method that uses common laboratory equipment (microcentrifuge) and spin cups. Through a series of extrusion and size exclusion steps, CDNs are produced from in vitro cell culture and are found to highly resemble the endogenous exosomes. Thus, we envision that this strategy holds great potential as a viable alternative to exosomes in the development of ideal DDS.

As extracellular vesicles (EVs) have become a prominent topic in life sciences, a growing number of studies are published on a regular basis addressing their biological relevance and possible applications. Nevertheless, the fundamental question of the true vesicular nature as well as possible influences on the EV secretion behavior have often been not adequately addressed. Furthermore, research regarding endothelial cell-derived EVs (EndoEVs) often focused on the large vesicular fractions comprising of microvesicles (MV) and apoptotic bodies. In this study we aimed to further extend the current knowledge of the influence of pre-isolation conditions, such as cell density and conditioning time, on EndoEV release from human umbilical vein endothelial cells (HUVECs). We combined fluorescence nanoparticle tracking analysis (NTA) and the established fluorescence-triggered flow cytometry (FT-FC) protocol to allow vesicle-specific detection and characterization of size and surface markers. We found significant effects of cell density and conditioning time on both abundance and size distribution of EndoEVs. Additionally, we present detailed information regarding the surface marker display on EVs from different fractions and size ranges. Our data provide crucial relevance for future projects aiming to elucidate EV secretion behavior of endothelial cells. Moreover, we show that the influence of different conditioning parameters on the nature of EndoEVs has to be considered.

Communication between dying cells and their environment is a critical process that promotes tissue homeostasis during normal cellular turnover, whilst during disease settings, it can contribute to inflammation through the release of intracellular factors. Extracellular vesicles (EVs) are a heterogeneous class of membrane-bound cell-derived structures that can engage in intercellular communication via the trafficking of bioactive molecules between cells and tissues. In addition to the well-described functions of EVs derived from living cells, the ability of dying cells to release EVs capable of mediating functions on target cells or tissues is also of significant interest. In particular, during inflammatory settings such as acute tissue injury, infection and autoimmunity, the EV-mediated transfer of proinflammatory cargo from dying cells is an important process that can elicit profound proinflammatory effects in recipient cells and tissues. Furthermore, the biogenesis of EVs via unique cell-death-associated pathways has also been recently described, highlighting an emerging niche in EV biology. This review outlines the mechanisms and functions of dying-cell-derived EVs and their ability to drive inflammation during various modes of cell death, whilst reflecting on the challenges and knowledge gaps in investigating this subgenre of extracellular vesicles research.

Morphological properties and the size of microvesicles were assessed using atomic force microscopy, electron microscopy, and granulometric analysis. As these methods require significant numbers of microvesicles, we chose microvesicles derived from cell lines for our research.

Improving outcomes for diffuse glioma patients requires methods that can accurately and sensitively monitor tumour activity and treatment response. Extracellular vesicles (EV) are membranous nanoparticles that can traverse the blood-brain-barrier, carrying oncogenic molecules into the circulation. Measuring clinically relevant glioma biomarkers cargoed in circulating EVs could revolutionise how glioma patients are managed. Despite their suitability for biomarker discovery, the co-isolation of highly abundant complex blood proteins has hindered comprehensive proteomic studies of circulating-EVs. Plasma-EVs isolated from pre-operative glioma grade II-IV patients ( n = 41) and controls ( n = 11) were sequenced by Sequential window acquisition of all theoretical fragment ion spectra mass spectrometry (SWATH-MS) and data extraction was performed by aligning against a custom 8662-protein library. Overall, 4054 proteins were measured in plasma-EVs. Differentially expressed proteins and putative circulating-EV markers were identified (adj. p -value < 0.05), including those reported in previous in-vitro and ex-vivo glioma-EV studies. Principal component analysis showed that plasma-EV protein profiles clustered according to glioma histological-subtype and grade, and plasma-EVs resampled from patients with recurrent tumour progression grouped with more aggressive glioma samples. The extensive plasma-EV proteome profiles achieved here highlight the potential for SWATH-MS to define circulating-EV biomarkers for objective blood-based measurements of glioma activity that could serve as ideal surrogate endpoints to assess tumour progression and allow more dynamic, patient-centred treatment protocols.

Circulating microparticles (MPs) are procoagulant due to the surface containing phosphatidylserine (PS), which facilitates coagulation. We investigated if MPs improve hemostasis in HA plasma models. MPs isolated from pooled normal human plasma were added to severe, moderate and mild HA plasma models (0%, 2.5%, 20% FVIII). The MPs' effect on hemostasis was evaluated by calibrated automated thrombogram (CAT) and overall hemostasis potential (OHP) assays, while fibrin structure was imaged by standard confocal, stimulated emission depletion (STED) microscopy and scanning electron microscopy (SEM). MPs partially restored thrombin generation and fibrin formation in all HA plasma models. The procoagulant effect of MPs requires PS exposure, to a less extent of contact pathway activation, but not tissue factor exposure or in vitro stimulation of MPs. MPs partially normalized the fibrin structure, and using super-resolution STED, MPs attached to fibrin were clearly resolved. In summary, our results demonstrate that PS positive MPs could improve hemostasis in HA plasma models.

Light luminescent microscopy was used to study the distribution of extracellular microvesicles with PKH26-stained membranes secreted by placenta-derived mesenchymal stromal cells in the uterine tissues at different terms after injections to intact rats and after abdominal delivery (a model of cesarian section). Microvesicles migrated through the uterine tissues and were detected for at least 8 days after injection. In some cases, microvesicles were more numerous in the uterus after cesarian section modeling, which can be related to blockade of microcirculation and lymph flow due to inflammation accompanying surgical intervention. The content of microvesicles in the uterine tissues gradually declined due to macrophage phagocytosis and, probably, due to their migration into the vascular bed. Despite their size, properly stained extracellular microvesicles can be detected by light microscopy in tissues after injections.

Microvesicles and exosomes are small membranous vesicles released to the extracellular environment and circulated throughout the body. Because they contain various parental cell-derived biomolecules such as DNA, mRNA, miRNA, proteins, and lipids, their enrichment and isolation are critical steps for their exploitation as potential biomarkers for clinical applications. However, conventional isolation methods (e.g., ultracentrifugation) cause significant loss and damage to microvesicles and exosomes. These methods also require multiple repetitive steps  of ultracentrifugation, loading, and wasting of reagents. This article describes a detailed method to fabricate an origami-paper-based device (Exo-PAD) designed for the effective enrichment and isolation of microvesicles and exosomes in a simple manner. The unique design of the Exo-PAD, consisting of accordion-like multifolded layers with convergent sample areas, is integrated with the ion concentration polarization technique, thereby enabling fivefold enrichment of the microvesicles and exosomes on specific layers. In addition, the enriched microvesicles and exosomes are isolated by simply unfolding the Exo-PAD.

Preeclampsia is the most common placental pathology in pregnant females, with increased morbidity and mortality incurred on the mother and the fetus. There is a need for improved biomarkers for diagnosis and monitoring of this condition. Placental syncytiotrophoblasts at the maternal-fetal interface release nanoparticles, including extracellular microvesicles, into the maternal blood during pregnancy. Syncytiotrophoblast extracellular microvesicles (STEVs) are being studied for their diagnostic potential and for their potential physiologic role in preeclampsia. We hypothesized that STEV profiles in maternal circulation would be altered under conditions of preeclampsia compared to normal pregnancy. Extracellular vesicles (EVs) released by BeWo cells in vitro showed high expression of syncytin-1, but no plac1 expression, demonstrating that trophoblast cell EVs express syncytin-1 on their surface. Placental alkaline phosphatase also showed high expression on BeWo EVs, but due to concern for cross reactivity to highly prevalent isoforms of intestinal and bone alkaline phosphatase, we utilized syncytin-1 as a marker for STEVs. In vivo, syncytin-1 protein expression was confirmed in maternal plasma EVs from Control and Preeclampsia subjects by Western blot, and overall, lower expression was noted in samples from patients with preeclampsia (n = 8). By nanoparticle analysis, EV profiles from Control and Preeclampsia groups showed similar total plasma EV quantities (p = 0.313) and size distribution (p = 0.415), but STEV quantitative signal, marked by syncytin-1 specific EVs, was significantly decreased in the Preeclampsia group (p = 2.8 × 10 -11 ). Receiver operating characteristic curve demonstrated that STEV signal threshold cut-off of <0.316 was 95.2% sensitive and 95.6% specific for diagnosis of preeclampsia in this cohort (area under curve = 0.975 ± 0.020). In conclusion, we report that the syncytin-1 expressing EV profiles in maternal plasma might serve as a placental tissue specific biomarker for preeclampsia.

Objective: Varicella zoster virus (VZV) can spread anterogradely and infect cerebral arteries causing VZV vasculopathy and arterial ischemic stroke. In this study, we tested the hypothesis that virus-infected cerebrovascular fibroblasts undergo phenotypic changes that promote vascular remodeling and facilitate virus transmission in an in vitro model of VZV vasculopathy. The aims of this project were therefore to examine the changes that virus-infected human brain adventitial vascular fibroblasts (HBVAFs) undergo in an in vitro model of VZV vasculopathy and to identify disease biomarkers relating to VZV-related vasculopathy., Methods: HBVAFs were infected with VZV, and their ability to migrate, proliferate, transdifferentiate, and interact with endothelial cells was studied with flow cytometry. Microparticles (MPs) released from these cells were isolated and imaged with transmission electron microscopy, and their protein content was analyzed with mass spectrometry. Circulating MP profiles were also studied in children with VZV and non-VZV vasculopathy and compared with controls., Results: VZV-infected HBVAFs transdifferentiated into myofibroblasts with enhanced proliferative and migratory capacity. Interaction of VZV-infected HBVAFs with endothelial cells resulted in endothelial dysfunction. These effects were, in part, mediated by the release of MPs from VZV-infected HBVAFs. These MPs contained VZV virions that could transmit VZV to neighboring cells, highlighting a novel model of VZV cell-to-cell viral dissemination. MPs positive for VZV were significantly higher in children with VZV-related vasculopathy compared to children with non-VZV vasculopathy ( p = 0.01) and controls ( p = 0.007)., Conclusions: VZV-infected HBVAFs promote vascular remodeling and facilitate virus transmission. These effects were mediated by the release of apoptotic MPs that could transmit VZV infection to neighboring cells through a Trojan horse means of productive viral infection. VZV+ MPs may represent a disease biomarker worthy of further study., (Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Novel therapeutic approaches that address the malignant cells in their stroma microenvironment are urgently needed in lung cancer. The stroma resident mesenchymal stem cells (MSCs) interact with cancer cells in diverse ways including microvesicles (MVs) that transfer proteins and RNA species thereby modulating recipient cells' phenotype. Previously, we have demonstrated that MSCs' secretome from the primary non-small cell lung cancer (NSCLC) niche (lung) and metastatic niche (bone marrow (BM)) demonstrate opposite effects on NSCLC cells in a translation initiation (TI) dependent manner. Here, we examined the effect of MVs secreted from BM-MSCs' or lung-MSCs (healthy, NSCLC) to NSCLC phenotype. Briefly, NSCLC cell lines treated with Lung or BM-MSCs' MVs were assayed for viability (WST-1), cell count/death (trypan), migration (scratch), TI status and MAPKs activation (immunoblotting). Corresponding to previous published trends, Lung-MSCs' MVs promoted NSCLC cells' assayed traits whereas, BM-MSCs' MVs suppressed them. Activation of MAPKs and autophagy was registered in lung-MSCs MVs treated NSCLC cell lines only. Furthermore, lung-MSCs' MVs' treated NSCLC cells demonstrated an early (5min) activation of MAPKs and TI factors (peIF4E/peIF4GI) not evident in BM-MSCs MVs treated cells. These observations depict a role for MSCs'-MVs in NSCLC phenotype design and display distinct differences between the primary and metastatic niches that correspond to disease progression. In conclusion, the systemic nature of MVs marks them as attractive therapeutic markers/targets and we propose that identification of specific cargoes/signals that differentiate between MSCs MVs of primary and metastatic niches may introduce fresh therapeutic approaches., (Copyright © 2019. Published by Elsevier Inc.)

Biological functions of extracellular vesicles (EVs) are being discovered to be critical in neurodegenerative disorders, including Parkinson's disease (PD). A previous study using cellular models of PD has suggested that EVs derived from microglia exposed to aggregated α-synuclein (α-Syn) leads to enhanced neurotoxicity. However, the function of EVs derived from microglia not treated with aggregated a-Syn or treated with monomeric α-Syn are unclear. Here, employing a widely used cellular model of PD, i.e. SH-SY5Y cells treated with MPP + , a well-established parkinsonian toxicant, we revealed that microglial EVs, when not stimulated by aggregated α-Syn, appeared to be protective, and the mechanisms, though remain to be defined further, appeared to involve mitochondrial dynamics, especially mitochondrial fission., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Leishmaniases, caused by Leishmania spp., are among the most prevalent infectious diseases in the world and their treatment may present high toxicity and side/adverse effects. This study evaluated the antileishmanial activity of the Hexanic Eluate subfraction from Maytenus guianensis bark (HEMg) incorporated in microparticles of PLGA. One batch of microparticles produced contained HEMg (HEMgP) and another contained the PLGA polymer alone (PCTE). The microparticles were characterized in regards to diameter, Zeta potential, encapsulation rate and morphology and their cytotoxicity was evaluated against J774 macrophages. The infection assay employing peritoneal macrophages witth L. amazonensis and cytokine dosages were performed on the cell supernatants. The groups of infected BALB/C mice were treated, euthanized and the parasite load and cytokine production were evaluated. The diameters and zeta potential were: 4 μm and -11.6 mV (PCTE) and 7.8 μm and -26.7 mV (HEMgP). The encapsulation rate was ≅ 15% and the morphology of the particles was spherical and homogeneous. In the infection assay, HEMgP inhibited the amastigotes by 70% (24 h) and 59% (48 h) and induced IL-12 and TNF-α production. HEMg in solution reduced the number of parasites in the lymph nodes by 50% and HEMgP administration increased the levels of IL-12 and TNF-α cytokines in lymph nodes and in the lesion site. When encapsulated, HEMg maintained its antileishmanial activity, but in a more attenuated and sustained form over time, showing promise as complementary/alternative therapy against cutaneous leishmaniasis., (Copyright © 2019 Elsevier Inc. All rights reserved.)

BACKGROUND Research on microparticles is rapidly evolving and has extended to the field of many diseases. It is unclear whether microparticles can be stored frozen. In this study, our goal was to verify whether cryopreservation had an effect on the properties of the microparticles. MATERIAL AND METHODS We obtained C57BL/6J mouse-derived microparticles by grinding and gradient centrifugation. The specimens were divided into 2 groups: without dimethyl sulfoxide and with dimethyl sulfoxide. The microparticles were then stored at 25°C, 4°C, -20°C, -80°C, and -196°C for 0.5 days, 1 day, 3 days, 5 days, and 7 days. We tested whether the concentration, coagulation function, diameter distribution, and morphology of the microparticles in the 2 groups changed compared to those of a fresh sample. RESULTS We discovered that the concentrations of total microparticles, annexin V-positive microparticles, and brain-derived microparticles changed with freezing. The coagulation function, morphology, and size distribution of the microparticles were also affected by cryopreservation. Finally, there was no difference in the effects of cryopreservation on microparticles between the dimethyl sulfoxide group and the dimethyl sulfoxide-free group. CONCLUSIONS This study suggests that cryopreservation has diverse effects on microparticles within 1 week and that dimethyl sulfoxide has no protective effect on cryopreserved microparticles. Therefore, microparticles should be used fresh for future studies, and they should not be cryopreserved with or without dimethyl sulfoxide.

Emerging evidence suggests that extracellular vesicle (EV)-containing miRNAs mediate intercellular communications in response to noxious stimuli. It remains unclear how a cell selectively sorts the cellular miRNAs into EVs. We report that caveolin-1 (cav-1) is essential for sorting of selected miRNAs into microvesicles (MVs), a main type of EVs generated by outward budding of the plasma membrane. We found that cav-1 tyrosine 14 (Y14)-phosphorylation leads to interactions between cav-1 and hnRNPA2B1, an RNA-binding protein. The cav-1/hnRNPA2B1 complex subsequently traffics together into MVs. Oxidative stress induces O -GlcNAcylation of hnRNPA2B1, resulting in a robustly altered hnRNPA2B1-bound miRNA repertoire. Notably, cav-1 pY14 also promotes hnRNPA2B1 O -GlcNAcylation. Functionally, macrophages serve as the principal recipient of epithelial MVs in the lung. MV-containing cav-1/hnRNPA2B1 complex-bound miR-17/93 activate tissue macrophages. Collectively, cav-1 is the first identified membranous protein that directly guides RNA-binding protein into EVs. Our work delineates a novel mechanism by which oxidative stress compels epithelial cells to package and secrete specific miRNAs and elicits an innate immune response., (© 2019 Lee et al.)

Microparticles are carriers of signals for intracellular signal transduction. These carriers include proteins, mRNAs, microRNAs and other bioactive substances. Platelets are a major source of circulating microparticles, and microparticles are closely associated with the development of certain cardiovascular diseases. In the present study, a method for separating, extracting and identifying platelet‑derived microparticles was developed and differences in the expression of surface proteins on microparticles harvested from platelets stimulated by vortexing or treatment with thrombin was investigated. The counts, composition, sizes and inner structures of microparticles were determined using flow cytometry and transmission electron microscopy. Additionally, it was demonstrated that platelets could be readily activated, and a large quantity of microparticles with varying complex compositions, structures and sizes were derived from activated platelets. High purity platelet‑derived microparticles were obtained by gradient centrifugation. However, the microparticles derived from platelets stimulated by thrombin treatment or vortexing differed significantly in the levels of CD63. The present study aimed to provide improved options for the extraction and identification of microparticles.

Trauma hemorrhage is a leading cause of death and disability worldwide. Platelets are fundamental to primary hemostasis, but become profoundly dysfunctional in critically injured patients by an unknown mechanism, contributing to an acute coagulopathy which exacerbates bleeding and increases mortality. The objective of this study was to elucidate the mechanism of platelet dysfunction in critically injured patients. We found that circulating platelets are transformed into procoagulant balloons within minutes of injury, accompanied by the release of large numbers of activated microparticles which coat leukocytes. Ballooning platelets were decorated with histone H4, a damage-associated molecular pattern released in massive quantities after severe injury, and exposure of healthy platelets to histone H4 recapitulated the changes in platelet structure and function observed in trauma patients. This is a report of platelet ballooning in human disease and of a previously unrecognized mechanism by which platelets contribute to the innate response to tissue damage., Competing Interests: The authors declare no conflict of interest., (Copyright © 2019 the Author(s). Published by PNAS.)

The endogenous micro/nano-particles in daily food have drawn much attention due to specific properties potential biological impact. The aim of this study was to investigate the nanoparticles in traditional fermented soybean sauces and vinegars in order to study the safety problems of nanoparticles in daily food. The transmission electron microscope results showed that all samples exhibited diverse nanostructures with diameters ranging from 10 to 400 nm. The concentration of nanoparticles in these foods was determined to be around 1.15 × 10 7 -3.43 × 10 9 particles/mL. Furthermore, the absorbance at 420 nm was found in all the fermented foods, which was ascribed to Maillard reaction products. The 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyl-tetrazolium bromide (MTT) results showed that nanoparticles in traditional fermented foods did not decrease cell viability in the concentration range tested (<200 μg/mL), which were equivalent to 20 L~200 L of selected soybean sauces and vinegars. However, further studies need to be performed to find out the interaction of nanoparticle with cell (food with body) after ingestion.

Microvesicles (MVs) derived from bone marrow niche components have an important role in genetic reprogramming and subsequent drugs induce apoptosis in leukemic cells. Here, we have found that undertreatment of curcumin or daunorubicin, the cross-talk through MVs of KG-1-bone marrow mesenchymal stem cells (BMSCs), significantly downregulates the expression of the survival gene osteopontin (OPN), CXCL-12, IL-6 (interleukin-6), STAT-3, and VCAM-1 (vascular cell adhesion molecule 1) in treated-KG-1 cells as well as exclusively upregulates CXCL-12 in BMSCs. Drug treated-cell populations' MVs of both single cultured osteoblasts (OBs) and cocultured KG-1 + BMSCs + OBs similarly upregulate survival mediators' OPN, CXCL-12, IL-6, STAT-3, and VCAM-1 in treated-KG-1 cells. Likewise, isolated MVs from KG-1 cells or communication between KG-1, BMSCs, and OBs treated by drugs increase the expression of genes OPN, CXCL-12, IL-6, STAT3, and VCAM-1 by OBs. MVs derived from KG-1 + BMSCs + OBs reduce drug-induced apoptosis in KG-1 cells. This suggests MVs-mediated information transfer is a procedure whereby OBs could overcome BMSCs-induced apoptosis in drug-treated-KG-1 cells., (© 2019 Wiley Periodicals, Inc.)

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival., Competing Interests: The authors declare no conflict of interest.

Weibel-Palade bodies (WPBs) are secretory granules that contain von Willebrand factor and P-selectin, molecules that regulate hemostasis and inflammation, respectively. The presence of CD63/LAMP3 in the limiting membrane of WPBs has led to their classification as lysosome-related organelles. Many lysosome-related organelles contain intraluminal vesicles (ILVs) enriched in CD63 that are secreted into the extracellular environment during cell activation to mediate intercellular communication. To date, there are no reports that WPBs contain or release ILVs. By light microscopy and live-cell imaging, we show that CD63 is enriched in microdomains within WPBs. Extracellular antibody recycling studies showed that CD63 in WPB microdomains can originate from the plasma membrane. By cryo-electron tomography of frozen-hydrated endothelial cells, we identify internal vesicles as novel structural features of the WPB lumen. By live-cell fluorescence microscopy, we directly observe the exocytotic release of EGFP-CD63 ILVs as discrete particles from individual WPBs. WPB exocytosis provides a novel route for release of ILVs during endothelial cell stimulation., (© 2019 by The American Society of Hematology.)

Platelet microparticles (PMPs) are released from the resting or activated platelet membrane, which includes a variety of proteins, mRNA, miRNA, lipids, and other substances. PMPs are involved in cell communication in vivo and are potential markers for multiple diseases. This narrative review focuses on the extraction, identification, and intercellular role of PMPs, with emphasis on the lesser known aspects of PMPs, namely, PMPs and complement systems, and their role in some diseases and disorders of women such as miscarriage and polycystic ovary syndrome, as well as the application of proteomics. For clinical purposes, we should first consider the importance of PMPs quantification and then what treatment should be given in response to increased quantities of PMPs as observed in many diseases. Currently, methods for quantification of PMPs are still in the experimental stages. Proteomics testing will expand biomarker discovery as well as future PMPs diagnostic and therapeutic applications., (© 2019 by the Association of Clinical Scientists, Inc.)

Fas-associated death domain (FADD) is a key adaptor molecule involved in numerous physiological processes including cell death, proliferation, innate immunity and inflammation. Therefore, changes in FADD expression have dramatic cellular consequences. In mice and humans, FADD regulation can occur through protein secretion. However, the molecular mechanisms accounting for human FADD secretion were still unknown. Here we report that canonical, non-canonical, but not alternative, NLRP3 inflammasome activation in human monocytes/macrophages induced FADD secretion. NLRP3 inflammasome activation by the bacterial toxin nigericin led to the proinflammatory interleukin-1β (IL-1β) release and to the induction of cell death by pyroptosis. However, we showed that FADD secretion could occur in absence of increased IL-1β release and pyroptosis and, reciprocally, that IL-1β release and pyroptosis could occur in absence of FADD secretion. Especially, FADD, but not IL-1β, secretion following NLRP3 inflammasome activation required extracellular glucose. Thus, FADD secretion was an active process distinct from unspecific release of proteins during pyroptosis. This FADD secretion process required K + efflux, NLRP3 sensor, ASC adaptor and CASPASE-1 molecule. Moreover, we identified FADD as a leaderless protein unconventionally secreted through microvesicle shedding, but not exosome release. Finally, we established human soluble FADD as a new marker of joint inflammation in gout and rheumatoid arthritis, two rheumatic diseases involving the NLRP3 inflammasome. Whether soluble FADD could be an actor in these diseases remains to be determined. Nevertheless, our results advance our understanding of the mechanisms contributing to the regulation of the FADD protein expression in human cells.

Cell membrane-derived microparticles (MPs), the critical mediators of intercellular communication, have gained much interest for use as natural drug delivery systems. Here, we examined the therapeutic potential of tumor cell-derived MPs (TMPs) in the context of malignant pleural effusion (MPE). TMPs packaging the chemotherapeutic drug methotrexate (TMPs-MTX) markedly restricted MPE growth and provided a survival benefit in MPE models induced by murine Lewis lung carcinoma and colon adenocarcinoma cells. On the basis of the potential benefit and minimal toxicity of TMPs-MTX, we conducted a human study of intrapleural delivery of a single dose of autologous TMPs packaging methotrexate (ATMPs-MTX) to assess their safety, immunogenicity, and clinical activity. We report our findings on 11 advanced lung cancer patients with MPE. We found that manufacturing and infusing ATMPs-MTX were feasible and safe, without evidence of toxic effects of grade 3 or higher. Evaluation of the tumor microenvironment in MPE demonstrated notable reductions in tumor cells and CD163 + macrophages in MPE after ATMP-MTX infusion, which then translated into objective clinical responses. Moreover, ATMP-MTX treatment stimulated CD4 + T cells to release IL-2 and CD8 + cells to release IFN-γ. Our initial experience with ATMPs-MTX in advanced lung cancer with MPE suggests that ATMPs targeting malignant cells and the immunosuppressive microenvironment may be a promising therapeutic platform for treating malignancies., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Background: Considering exosomes as intercellular transporters, inevitably interacting with the plasma membrane and the large available surface of blood cells, we wonder if a fraction of circulating exosomes is associated with the surface of blood cells., Objective: The aim of this study was to develop an efficient protocol for isolating exosomes associated with the surface of blood cells and to further investigate the characteristics of this fraction in a healthy state and during the development of breast cancer, as well as its possible implication for use in diagnostic applications., Methods: Blood samples were collected from Healthy Females (HFs) and breast cancer patients (BCPs). Exosomes extracted from blood plasma and eluted from the surface of blood cells were isolated by ultrafiltration with subsequent ultracentrifugation., Results: Transmission Electron Microscopy (TEM), along with immunogold labeling, demonstrated the presence of exosomes among membrane-wrapped extracellular vesicles (EVs) isolated from both plasma and blood cell eluates. TEM, nanoparticle tracking analysis, and NanoOrange protein quantitation data showed that cell-associated exosomes constituted no less than 2/3 of total blood exosome number. Exosomes, ranging from 50-70 nm in size, prevailed in the blood of breast cancer patients, whereas smaller exosomes (30-50 nm) were mostly observed in the blood of healthy women. Analysis of specific proteins and RNAs in exosomes circulating in blood demonstrated the significant differences in the packing density of the polymers in exosomes of HFs and BCPs. Preliminary data indicated that detection of cancer-specific miRNA (miR-103, miR-191, miR-195) in exosomes associated with the fraction of red blood cells allowed to discriminate HFs and BCPs more precisely compared to cell-free exosomes circulating in plasma., Conclusion: Our data provide the basis for using blood cell-associated exosomes for diagnostic applications., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Extracellular vesicles (EVs) have prevalent roles in cancer biology and regenerative medicine. Conventional techniques for characterising EVs including electron microscopy (EM), nanoparticle tracking analysis (NTA) and tuneable resistive pulse sensing (TRPS), have been reported to produce high variability in particle count (EM) and poor sensitivity in detecting EVs below 50 nm in size (NTA and TRPS), making accurate and unbiased EV analysis technically challenging. This study introduces direct stochastic optical reconstruction microscopy (d-STORM) as an efficient and reliable characterisation approach for stem cell-derived EVs. Using a photo-switchable lipid dye, d-STORM imaging enabled rapid detection of EVs down to 20-30 nm in size with higher sensitivity and lower variability compared to EM, NTA and TRPS techniques. Imaging of EV uptake by live stem cells in culture further confirmed the potential of this approach for downstream cell biology applications and for the analysis of vesicle-based cell-cell communication., (Copyright © 2018 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: A strong correlation exists between type 2 diabetes mellitus (T2DM) and cardiovascular disease (CVD), with CVD and the presence of atherosclerosis being the prevailing cause of morbidity and mortality in diabetic populations. T2DM is accompanied by various coagulopathies, including anomalous clot formation or amyloid fibrin(ogen), the presence of dysregulated inflammatory molecules. Platelets are intimately involved in thrombus formation and particularly vulnerable to inflammatory cytokines., Methods: The aim of this current study was therefore to assess whole blood (hyper)coagulability, platelet ultrastructure and receptor expression, as well as the levels of IL-1β, IL-6, IL-8 and sP-selectin in healthy and diabetic individuals. Platelet morphology was assessed through scanning electron microscopy (SEM), while assessment of GPIIb/IIIa receptor expression was performed with confocal microscopy and flow cytometry with the addition of FITC-PAC-1 and CD41-PE antibodies. IL-1β, IL-6 and IL-8 and sP-selectin levels were assessed using a multiplex assay., Results: In T2DM there is significant upregulation of circulating inflammatory markers, hypercoagulation and platelet activation, with increased GPIIb/IIIa receptor expression, as seen with flow cytometry and confocal microscopy. Analyses showed that these receptors were additionally shed onto microparticles, which was confirmed with SEM., Conclusions: Cumulatively, this provides mechanistic evidence that pathological states of platelets together with amyloid fibrin(ogen) in T2DM, might underpin an increased risk for cardiovascular events.

Macrophages are generally assumed to unload surplus cholesterol through direct interactions between ABC transporters on the plasma membrane and HDLs, but they have also been reported to release cholesterol-containing particles. How macrophage-derived particles are formed and released has not been clear. To understand the genesis of macrophage-derived particles, we imaged mouse macrophages by EM and nanoscale secondary ion mass spectrometry (nanoSIMS). By scanning EM, we found that large numbers of 20- to 120-nm particles are released from the fingerlike projections (filopodia) of macrophages. These particles attach to the substrate, forming a "lawn" of particles surrounding macrophages. By nanoSIMS imaging we showed that these particles are enriched in the mobile and metabolically active accessible pool of cholesterol (detectable by ALO-D4, a modified version of a cholesterol-binding cytolysin). The cholesterol content of macrophage-derived particles was increased by loading the cells with cholesterol or by adding LXR and RXR agonists to the cell-culture medium. Incubating macrophages with HDL reduced the cholesterol content of macrophage-derived particles. We propose that release of accessible cholesterol-rich particles from the macrophage plasma membrane could assist in disposing of surplus cholesterol and increase the efficiency of cholesterol movement to HDL., Competing Interests: The authors declare no conflict of interest.

Aims: Previous studies have demonstrated improved cardiac function following myocardial infarction (MI) after administration of endothelial progenitor cells (EPCs) into ischaemic myocardium. A growing body of literature supports paracrine effectors, including extracellular vesicles (EVs), as the main mediators of the therapeutic benefits of EPCs. The direct use of paracrine factors is an attractive strategy that harnesses the effects of cell therapy without concerns of cell engraftment or viability. We aim to reproduce the beneficial effects of EPC treatment through delivery of EPC-derived EVs within a shear-thinning gel (STG) for precise localization and sustained delivery., Methods and Results: EVs were harvested from EPCs isolated from adult male Rattus norvegicus (Wistar) rats and characterized by electron microscopy, nanoparticle tracking analysis (NTA), and mass spectrometry. EVs were incorporated into the STG and injected at the border zone in rat models of MI. Haemodynamic function, angiogenesis, and myocardial remodelling were analyzed in five groups: phosphate buffered saline (PBS) control, STG control, EVs in PBS, EVs in STG, and EPCs in STG. Electron microscopy and NTA of EVs showed uniform particles of 50-200 nm. EV content analysis revealed several key angiogenic mediators. EV uptake by endothelial cells was confirmed and followed by robust therapeutic angiogenesis. In vivo animal experiments demonstrated that delivery of EVs within the STG resulted in increased peri-infarct vascular proliferation, preservation of ventricular geometry, and improved haemodynamic function post-MI., Conclusions: EPC-derived EVs delivered into ischaemic myocardium via an injectable hydrogel enhanced peri-infarct angiogenesis and myocardial haemodynamics in a rat model of MI. The STG greatly increased therapeutic efficiency and efficacy of EV-mediated myocardial preservation.

Staphylococcus aureus membrane vesicles (MVs) aggravate atopic dermatitis (AD) through the delivery of bacterial effector molecules to host cells and the stimulation of inflammatory responses. This study investigated the inhibitory effect of thymol, a phenolic monoterpene found in essential oils derived from plants, on the worsening of AD induced by S. aureus MVs both in vitro and in vivo. The sub-minimal inhibitory concentrations of thymol disrupted S. aureus MVs. Intact S. aureus MVs induced the expression of pro-inflammatory cytokine (interleukin (IL)-1β, IL-6, and tumor necrosis factor-α) and chemokine (IL-8 and monocyte chemoattractant protein-1) genes in cultured keratinocytes, whereas thymol-treated S. aureus MVs did not stimulate the expression of these genes. Topical application of thymol-treated S. aureus MVs or treatment with thymol after intact S. aureus MVs to AD-like skin lesions diminished the pathology of AD. This included decreases in epidermal/dermal thickness and infiltration of eosinophils/mast cells, and inhibited expression of pro-inflammatory cytokine and chemokine genes in mouse AD model. Moreover, thymol significantly suppressed the Th1, Th2, and Th17-mediated inflammatory responses in AD-like skin lesions induced by S. aureus MVs, and reduced the serum levels of immunoglobulin (Ig) G2a, mite-specific IgE, and total IgE. In summary, thymol disrupts S. aureus MVs and suppresses inflammatory responses in AD-like skin lesions aggravated by S. aureus MVs. Our results suggest that thymol is a possible candidate for the management of AD aggravation induced by S. aureus colonization or infection in the lesions., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Extracellular vesicles are a heterogeneous group of cell-derived membranous structures comprising exosomes and microvesicles, which originate from the endosomal system or which are shed from the plasma membrane, respectively. They are present in biological fluids and are involved in multiple physiological and pathological processes. Extracellular vesicles are now considered as an additional mechanism for intercellular communication, allowing cells to exchange proteins, lipids and genetic material. Knowledge of the cellular processes that govern extracellular vesicle biology is essential to shed light on the physiological and pathological functions of these vesicles as well as on clinical applications involving their use and/or analysis. However, in this expanding field, much remains unknown regarding the origin, biogenesis, secretion, targeting and fate of these vesicles.

A number of neuronal and glial proteins were previously found to be released in free-standing soluble form from cultured brain cells into cell-conditioned media. Here, we sought to examine if similar proteins are also contained in neural and astroglial cell-released extracellular microvesicles/exosomes (MV/E). In this study, MV/E were isolated from cell-conditioned media from control and cytotoxin-challenged rat cerebrocortical mixed culture (CTX) and mouse neuroblastoma N2a cells. Cytotoxin challenges included pro-necrosis calcium ionophore A23187, pro-apoptosis staurosporine (STS), and excitotoxin N-methyl-D-aspartate. Based on established nanoparticle characterization method (dynamic light scattering, NanoTracker, and transmission electron microscopy), we confirmed that these released vesicles are in fact characteristic representation of MV/E by morphology (lipid bilayered vesicles) and by particle size (132-142 nm for CTX and 49-77 nm for N2a cells). We indeed identified neural cell body protein UCH-L1, axonal injury marker αII-spectrin and its breakdown products (SBDPs), astroglial markers GFAP and its breakdown products (GFAP-BDP), dendritic protein BIII-tubulin, synaptic protein synaptophysin, and exosome marker Alix in microvesicles from CTX and/or N2a cells. Furthermore, SBDPs, GFAP-BDP, UCH-L1, and synaptophysin are especially dominant in MV/E isolated from cytotoxin-treated CTX cells. Similarly, SBDPs, βIII-tubulin, and UCH-L1 are more prominently observed in cytotoxin-challenged N2a cells. Lastly, when isolated MV/E from A23187- or STS-challenged N2a cells were introduced to healthy N2a culture, they are capable of evoking cytotoxicity in the latter. Taken together, our study identified that microvesicles/exosomes isolated form healthy and injured brain cells contain certain neural and astroglial proteins, as well as possibly other cytotoxic factors that are capable of propagating cytotoxic effects.

Objectives: Mesenchymal stem cells (MSCs) release extracellular vesicles (EVs) that display a therapeutic effect in inflammatory disease models. Although MSCs can prevent arthritis, the role of MSCs-derived EVs has never been reported in rheumatoid arthritis. This prompted us to compare the function of exosomes (Exos) and microparticles (MPs) isolated from MSCs and investigate their immunomodulatory function in arthritis. Methods: MSCs-derived Exos and MPs were isolated by differential ultracentrifugation. Immunosuppressive effects of MPs or Exos were investigated on T and B lymphocytes in vitro and in the Delayed-Type Hypersensitivity (DTH) and Collagen-Induced Arthritis (CIA) models. Results: Exos and MPs from MSCs inhibited T lymphocyte proliferation in a dose-dependent manner and decreased the percentage of CD4 + and CD8 + T cell subsets. Interestingly, Exos increased Treg cell populations while parental MSCs did not. Conversely, plasmablast differentiation was reduced to a similar extent by MSCs, Exos or MPs. IFN-γ priming of MSCs before vesicles isolation did not influence the immunomodulatory function of isolated Exos or MPs. In DTH, we observed a dose-dependent anti-inflammatory effect of MPs and Exos, while in the CIA model, Exos efficiently decreased clinical signs of inflammation. The beneficial effect of Exos was associated with fewer plasmablasts and more Breg-like cells in lymph nodes. Conclusions: Both MSCs-derived MPs and Exos exerted an anti-inflammatory role on T and B lymphocytes independently of MSCs priming. However, Exos were more efficient in suppressing inflammation in vivo . Our work is the first demonstration of the therapeutic potential of MSCs-derived EVs in inflammatory arthritis., Competing Interests: Competing Interest: The authors have no financial or personal conflict of interest to disclose.

Sepsis is associated with dysfunctional coagulation. Recent data suggest that platelets play a role in sepsis by promoting neutrophil accumulation. Herein, we show that cecal ligation and puncture (CLP) triggered systemic inflammation, which is characterized by formation of IL-6 and CXC chemokines as well as neutrophil accumulation in the lung. Platelet depletion decreased neutrophil accumulation, IL-6, and CXC chemokines formation in septic lungs. Depletion of platelets increased peak thrombin formation and total thrombin generation (TG) in plasma from septic animals. CLP elevated circulating levels of platelet-derived microparticles (PMPs). In vitro generated PMPs were a potent inducer of TG. Interestingly, in vitro wild-type recombinant annexin V abolished PMP-induced thrombin formation whereas a mutant annexin V protein, which does not bind to phosphatidylserine (PS), had no effect. Administration of wild-type, but not mutant annexin V, significantly inhibited thrombin formation in septic animals. Moreover, CLP-induced formation of thrombin-antithrombin complexes were reduced in platelet-depleted mice and in animals pretreated with annexin V. PMP-induced TG attenuated in FXII- and FVII-deficient plasma. These findings suggest that sepsis-induced TG is dependent on platelets. Moreover, PMPs formed in sepsis are a potent inducer of TG via PS exposure, and activation of both the intrinsic and extrinsic pathway of coagulation. In conclusion, these observations suggest that PMPs and PS play an important role in dysfunctional coagulation in abdominal sepsis., (© 2017 Wiley Periodicals, Inc.)

Mesenchymal stromal cells (MSC) are a promising therapy for immunological disorders. However, culture expanded MSC are large and get trapped in the capillary networks of the lungs after intravenous infusion, where they have a short survival time. Hypothetically, living cells are a risk for tumor formation. To reduce risks associated with MSC infusion and improve the distribution in the body, we generated membrane particles (MP) of MSC and MSC stimulated with IFN-γ (MPγ). Tracking analysis and electron microscopy indicated that the average size of MP was 120 nm, and they showed a round shape. MP exhibited ATPase, nucleotidase and esterase activity, indicating they are enzymatically active. MP and MPγ did not physically interact with T cells and had no effect on CD4 + and CD8 + T cells proliferation. However, MP and MPγ selectively bound to monocytes and decreased the frequency of pro-inflammatory CD14 + CD16 + monocytes by induction of selective apoptosis. MP and MPγ increased the percentage of CD90 positive monocytes, and MPγ but not MP increased the percentage of anti-inflammatory PD-L1 monocytes. MPγ increased mRNA expression of PD-L1 in monocytes. These data demonstrate that MP have immunomodulatory properties and have potential as a novel cell-free therapy for treatment of immunological disorders.

Microparticles (MPs) are cell-cell communication vesicles derived from the cell surface plasma membrane, although they are not known to originate from cardiac ventricular muscle. In ventricular cardiomyocytes, the membrane deformation protein cardiac bridging integrator 1 (cBIN1 or BIN1+13+17) creates transverse-tubule (t-tubule) membrane microfolds, which facilitate ion channel trafficking and modulate local ionic concentrations. The microfold-generated microdomains continuously reorganize, adapting in response to stress to modulate the calcium signaling apparatus. We explored the possibility that cBIN1-microfolds are externally released from cardiomyocytes. Using electron microscopy imaging with immunogold labeling, we found in mouse plasma that cBIN1 exists in membrane vesicles about 200 nm in size, which is consistent with the size of MPs. In mice with cardiac-specific heterozygous Bin1 deletion, flow cytometry identified 47% less cBIN1-MPs in plasma, supporting cardiac origin. Cardiac release was also evidenced by the detection of cBIN1-MPs in medium bathing a pure population of isolated adult mouse cardiomyocytes. In human plasma, osmotic shock increased cBIN1 detection by enzyme-linked immunosorbent assay (ELISA), and cBIN1 level decreased in humans with heart failure, a condition with reduced cardiac muscle cBIN1, both of which support cBIN1 release in MPs from human hearts. Exploring putative mechanisms of MP release, we found that the membrane fission complex endosomal sorting complexes required for transport (ESCRT)-III subunit charged multivesicular body protein 4B (CHMP4B) colocalizes and coimmunoprecipitates with cBIN1, an interaction enhanced by actin stabilization. In HeLa cells with cBIN1 overexpression, knockdown of CHMP4B reduced the release of cBIN1-MPs. Using truncation mutants, we identified that the N-terminal BAR (N-BAR) domain in cBIN1 is required for CHMP4B binding and MP release. This study links the BAR protein superfamily to the ESCRT pathway for MP biogenesis in mammalian cardiac ventricular cells, identifying elements of a pathway by which cytoplasmic cBIN1 is released into blood.

The effect of conditioned medium (CM) or microvesicles (MVs), secreted by multicellular spheroids of oviductal cells, and the involvement of some microRNAs (miRNAs) were investigated in canine oocyte maturation. To generate CM, spheroids were cultured for 3 days. MVs were obtained by ultracentrifugation of CM at 100,000  g and measured for size and concentration by NanoSight instrument. Cumulus-oocyte complexes (COCs) were matured at 38.5°C with 5% CO 2 and 5% of O 2 in synthetic oviductal fluid (SOF) in biphasic systems: for 24 h, with 5.0 μg/mL of LH and for other 48 h with 10% oestrous bitch serum. SOF was used as control (CTR) or supplemented with 10% CM or 25-50-75-100-150 × 10 6  MVs/mL labeled with PKH-26. Results show that multicellular aggregates secreted shedding vesicles. By fluorescence microscopy, the incorporation of labeled MVs was visible only at 72 h in oocyte cytoplasm. These MVs had a positive effect ( P  < 0.05) on maturation rate (MII) at the concentration of 75 and 100 × 10 6  MVs/mL compared to CM and CTR (20.34% and 21.82% vs 9.09% and 8.66% respectively). The concentration of 150 × 10 6  MVs/mL provided only 9.26% of MII. The expression of three specific miRNAs (miR-30b, miR-375 and miR-503) was studied. The lower rate of MII with the higher concentration of MVs is possibly due to the high level of miR-375. In conclusion, the oviductal MVs could be involved in cellular trafficking during oocyte maturation and their possible use in vitro could facilitate the exploitment of canine reproductive biotechnologies., (© 2017 Society for Reproduction and Fertility.)

Essentials Platelet microparticles play a major role in pathologies, including hemostasis and thrombosis. Platelet microparticles have been analyzed and classified based on their ultrastructure. The structure and intracellular origin of microparticles depend on the cell-activating stimulus. Thrombin-treated platelets fall apart and form microparticles that contain cellular organelles., Summary: Background Platelet-derived microparticles comprise the major population of circulating blood microparticles that play an important role in hemostasis and thrombosis. Despite numerous studies on the (patho)physiological roles of platelet-derived microparticles, mechanisms of their formation and structural details remain largely unknown. Objectives Here we studied the formation, ultrastructure and composition of platelet-derived microparticles from isolated human platelets, either quiescent or stimulated with one of the following activators: arachidonic acid, ADP, collagen, thrombin or calcium ionophore A23187. Methods Using flow cytometry, transmission and scanning electron microscopy, we analyzed the intracellular origin, structural diversity and size distributions of the subcellular particles released from platelets. Results The structure, dimensions and intracellular origin of microparticles depend on the cell-activating stimulus. The main structural groups include a vesicle surrounded by one thin membrane or multivesicular structures. Thrombin, unlike other stimuli, induced formation of microparticles not only from the platelet plasma membrane and cytoplasm but also from intracellular structures. A fraction of these vesicular particles having an intracellular origin contained organelles, such as mitochondria, glycogen granules and vacuoles. The size of platelet-derived microparticles depended on the nature of the cell-activating stimulus. Conclusion The results obtained provide a structural basis for the qualitative differences of various platelet activators, for specific physiological and pathological effects of microparticles, and for development of advanced assays., (© 2017 International Society on Thrombosis and Haemostasis.)

Blood microparticles (MPs) are small membrane vesicles (50-1000nm), derived from different cell types. They are known to play important roles in various biological processes and also recognized as potential biomarkers of various health disorders. Different methods are currently used for the detection and characterization of MPs, but none of these methods is capable to quantify and qualify total MPs at the same time, hence, there is a need to develop a new approach for simultaneous detection, characterization and quantification of microparticles. Here we show the potential of surface plasmon resonance (SPR) method coupled to atomic force microscopy (AFM) to quantify and qualify platelet-derived microparticles (PMPs), on the whole nano-to micro-meter scale. The different subpopulations of microparticles could be determined via their capture onto the surface using specific ligands. In order to verify the correlation between the capture level and the microparticles concentration in solution, two calibration standards were used: Virus-Like Particles (VLPs) and synthetic beads with a mean diameter of 53nm and 920nm respectively. The AFM analysis of the biochip surface allowed metrological analysis of captured PMPs and revealed that more than 95% of PMPs were smaller than 300nm. Our results suggest that our NanoBioAnalytical platform, combining SPR and AFM, is a suitable method for a sensitive, reproducible, label-free characterization and quantification of MPs over a wide concentration range (≈10 7 to 10 12 particles/mL; with a limit of detection (LOD) in the lowest ng/µL range) which matches with their typical concentrations in blood., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Previous studies have concluded that microparticles (MPs) can more effectively approach the microvessel wall than nanoparticles because of margination. In this study, however, we show that MPs are not marginated in capillaries where the vessel diameter is comparable to that of red blood cells (RBCs). We numerically investigated the behavior of MPs with a diameter of 1 μm in various microvessel sizes, including capillaries. In capillaries, the flow mode of RBCs shifted from multi-file flow to bolus (single-file) flow, and MPs were captured by the bolus flow of the RBCs instead of being marginated. Once MPs were captured, they rarely escaped from the vortex-like flow structures between RBCs. These capture events were enhanced when the hematocrit was decreased, and reduced when the shear rate was increased. Our results suggest that microparticles may be rather inefficient drug carriers when targeting capillaries because of capture events, but nanoparticles, which are more randomly distributed in capillaries, may be more effective carriers.

During the pregnancy associated syndrome preeclampsia (PE), there is increased release of placental syncytiotrophoblast extracellular vesicles (STBEVs) and free foetal haemoglobin (HbF) into the maternal circulation. In the present study we investigated the uptake of normal and PE STBEVs by primary human coronary artery endothelial cells (HCAEC) and the effects of free HbF on this uptake. Our results show internalization of STBEVs into primary HCAEC, and transfer of placenta specific miRNAs from STBEVs into the endoplasmic reticulum and mitochondria of these recipient cells. Further, the transferred miRNAs were functional, causing a down regulation of specific target genes, including the PE associated gene fms related tyrosine kinase 1 (FLT1). When co-treating normal STBEVs with HbF, the miRNA deposition is altered from the mitochondria to the ER and the cell membrane becomes ruffled, as was also seen with PE STBEVs. These findings suggest that STBEVs may cause endothelial damage and contribute to the endothelial dysfunction typical for PE. The miRNA mediated effects on gene expression may contribute to the oxidative and endoplasmic reticulum stress described in PE, as well as endothelial reprogramming that may underlay the increased risk of cardiovascular disease reported for women with PE later in life.