Helium Conditioning Increases Cardiac Fibroblast Migration Which Effect Is Not Propagated via Soluble Factors or Extracellular Vesicles.

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% CO2 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.

Telocytes in the human ascending aorta: Characterization and exosome-related KLF-4/VEGF-A expression.

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-ß, CD29/integrin ß-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.

HTNV infection of CD8+ T cells is associated with disease progression in HFRS patients.

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.

Isolation of Small Extracellular Vesicles from Human Sera.

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.

Enumeration and phenotyping of circulating microvesicles by flow cytometry and nanoparticle tracking analysis: Plasma versus serum.

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.

Cell-Derived Nanovesicles as Exosome-Mimetics for Drug Delivery Purposes: Uses and Recommendations.

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.

Elongated neutrophil-derived structures are blood-borne microparticles formed by rolling neutrophils during sepsis.

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.

Fluorescence-Based Nanoparticle Tracking Analysis and Flow Cytometry for Characterization of Endothelial Extracellular Vesicle Release.

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.

Stoking the Fire: How Dying Cells Propagate Inflammatory Signalling through Extracellular Vesicle Trafficking.

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.

Methodological Approaches to Assessing the Size and Morphology of Microvesicles of Cell Lines.

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.

A Comprehensive Proteomic SWATH-MS Workflow for Profiling Blood Extracellular Vesicles: A New Avenue for Glioma Tumour Surveillance.

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.

Preservation of Mesenchymal Stem Cell-Derived Extracellular Vesicles after Abdominal Delivery in the Experiment.

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.

Paper-Based Preconcentration and Isolation of Microvesicles and Exosomes.

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.

Phosphatidylserine positive microparticles improve hemostasis in in-vitro hemophilia A plasma models.

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.

Syncytiotrophoblast extracellular microvesicle profiles in maternal circulation for noninvasive diagnosis of preeclampsia.

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.

Microparticle-mediated VZV propagation and endothelial activation: Mechanism of VZV vasculopathy.

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.

Niche origin of mesenchymal stem cells derived microvesicles determines opposing effects on NSCLC: Primary versus metastatic.

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.

Effects of Cryopreservation on Microparticles Concentration, Procoagulant Function, Size Distribution, and Morphology.

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.

Extracellular microvesicles-derived from microglia treated with unaggregated α-synuclein attenuate mitochondrial fission and toxicity-induced by Parkinsonian toxin MPP.

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.