MAM-STAT3-induced upregulation of mitochondrial Ca+2 causes immunosenescence in patients with type A mandibuloacral dysplasia

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Abstract Homozygous lamina/c p.R527C mutations result in severe mandibuloacral dysplasia (MAD) and progeroid syndrome, but the underlying molecular pathology remains unknown. Here, we report on three patients with MAD, all displaying severe systemic inflammaging and characterized the major molecular pathways involved in the manifestation of this disease. Analysis of induced pluripotent stem cell (IPSC)-derived mesenchymal stem cells (MAD-iMSCs) obtained from the patients revealed that increased mitochondrial Ca +2 loading was the root cause of lost mitochondrial membrane potential, abnormal fission/fusion and fragmentation, which then participated in inflammaging by inducing the inflammasome. These alterations in Ca +2 homeostasis were mediated by signal transducer and activator of transcription 3 (STAT3), which is located on the mitochondrial associated membrane (MAM). STAT3 function could be rescued by treatment with clinically-approved IL-6 blockers, or by correction of R527C mutations. In addition, extracellular vesicles (EVs) obtained from MAD-iMSCs displayed reduced immunomodulatory function, being unable to rescue bleomycin-induced lung fibrosis and triggering mitochondrial dysfunction, senescence, and fibrosis in healthy cells. Our results provide new insights into the pathology of complex lamin-associated MAD with systemic immunosenescence, and suggest that targeting defective mitochondrial Ca +2 homeostasis may represent a promising novel therapy for this condition.
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Urinary Extracellular Vesicles and Their miRNA Cargo in Patients with Fabry Nephropathy
Current biomarkers of Fabry nephropathy lack sensitivity in detecting early kidney damage and do not predict progression of nephropathy. Urinary extracellular vesicles (uEVs) and their molecular cargo could reflect early changes in renal impairment as they are secreted by the cells lining the urinary tract. We aimed to conduct a proof-of-concept study to investigate whether analysis of uEV characteristics and expression of uEV-derived microRNAs (miRNAs) could be applicable in studies to predict the development and progression of nephropathy in Fabry disease. A total of 20 Fabry patients were divided into two groups, depending on the presence of nephropathy. Chronological urine samples collected during 10-year follow-up were used for uEVs isolation with size exclusion chromatography. Nanoparticle tracking analysis was used to determine concentration and size of uEVs. We evaluated the expression of five uEV-derived miRNAs by qPCR (miR-23a-3p, miR-29a-3p, miR-30b-5p, miR-34a-5p, miR-200a-3p). There was no difference in the concentration and size of uEVs between patients with and without nephropathy at last follow-up or longitudinally. However, we found increased expression of miR-29a-3p and miR-200a-3p in uEVs isolated from chronological samples of patients with Fabry nephropathy. This may indicate an attempt by the organism to prevent the progression of renal damage leading to end-stage renal disease as previously reported in type 1 diabetes. In addition, we found an increased expression of miR-30b-5p in the 10-year period in uEVs of patients without renal dysfunction. miR-30b-5 was reported to have a protective role in podocyte injury and may possibly be important in Fabry nephropathy. These findings indicate that uEVs and their molecular cargo could be a promising target of studies focusing on elucidation of Fabry nephropathy. Nevertheless, total concentration and size of uEVs were neither indicative of the presence nor progression of Fabry nephropathy, while the role of the analyzed miRNAs in Fabry nephropathy progression was merely indicated and needs further in-depth studies.
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2022
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Proteomics Analyses Reveal Functional Differences between Exosomes of Mesenchymal Stem Cells Derived from The Umbilical Cord and Those Derived from The Adipose Tissue
OBJECTIVE: We aimed to identify the differentially expressed proteins (DEPs) and functional differences between exosomes derived from mesenchymal stem cells (MSCs) derived from umbilical cord (UC) or adipose tissue (AD). MATERIALS AND METHODS: In this experimental study, the UC and AD were isolated from healthy volunteers. Then, exosomes from UC-MSCs and AD-MSCs were isolated and characterized. Next, the protein compositions of the exosomes were examined via liquid chromatography tandem mass spectrometry (LC-MS/MS), followed by evaluation of the DEPs between UC-MSC and AD-MSC-derived exosomes. Finally, functional enrichment analysis was performed. RESULTS: One hundred and ninety-eight key DEPs were identified, among which, albumin (ALB), alpha-II-spectrin (SPTAN1), and Ras-related C3 botulinum toxin substrate 2 (RAC2) were the three hub proteins present at the highest levels in the protein-protein interaction network that was generated based on the shared DEPs. The DEPs were mainly enriched in gene ontology (GO) items associated with immunity, complement activation, and protein activation cascade regulation corresponding to 24 pathways, of which complement and coagulation cascades as well as platelet activation pathways were the most significant. CONCLUSION: The different functions of AD- and UC-MSC exosomes in clinical applications may be related to the differences in their immunomodulatory activities.
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2022
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INTRODUCTION/PURPOSE: High-intensity interval training (HIIT) promotes various biological processes and metabolic effects in multiple organs, but the role of extracellular vesicles (EVs) released from a variety of cells is not fully understood during HIIT exercise (HIIT-Ex). We investigated the changes in circulating number and proteomic profile of EVs to assess the effect of HIIT-Ex. METHODS: Seventeen young men (median age, 20 years) were enrolled in the study. Total duration of the HIIT-Ex was 4 min. Blood samples were collected from before HIIT-Ex (pre-HIIT-Ex), at the immediate conclusion of HIIT-Ex (T0), at 30 min (T30), and at 120 min after HIIT-Ex. The pulse rate and systolic blood pressure were measured. Circulating EVs were characterized, and EV proteins were detected via nano liquid chromatography tandem mass spectrometry. RESULTS: The pulse rate and systolic blood pressure at T0 to pre-HIIT-Ex were significantly higher. Circulating EV number was significantly altered throughout the HIIT-Ex, and the source of circulating EVs included skeletal muscle, hepatocytes, and adipose tissue. Proteomic analysis identified a total of 558 proteins within isolated circulating EVs from pre-HIIT-Ex, T0, and T30. Twenty proteins in total were significantly changed at pre-HIIT-Ex, T0, and T30 and are involved in a variety of pathways, such as activation of coagulation cascades, cellular oxidant detoxification, and correction of acid-base imbalance. Catalase and peroxiredoxin II were increased at T0. CONCLUSION: The circulating EV composition can be immediately changed by particularly a short time of HIIT-Ex, indicating that EVs may intercommunicate across various organs rapidly in response to HIIT-Ex.
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2022
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Inhibition of neutral sphingomyelinase 2 reduces extracellular vesicle release from neurons, oligodendrocytes, and activated microglial cells following acute brain injury
Extracellular Vesicles (EVs) are implicated in the spread of pathogenic proteinsin a growing number of neurological diseases. Given this, there is rising interest in developing inhibitors of Neutral Sphingomyelinase 2 (nSMase2), an enzyme critical in EV biogenesis. Our group recently discovered phenyl(R)-(1-(3-(3,4-dimethoxyphenyl)-2,6-dimethylimidazo[1,2-b]pyridazin-8-yl)pyrrolidin-3-yl)carbamate (PDDC), the first potent, selective, orally-available, and brain-penetrable nSMase2 inhibitor, capable of dose-dependently reducing EVs release in vitro and in vivo. Herein, using multiplexed Surface Plasmon Resonance imaging (SPRi), we evaluated which brain cell-derived EVs were affected by PDDC following acute brain injury. Mice were fed PDDC-containing chow at doses which gave steady PDDC brain exposures exceeding its nSMase2 IC50. Mice were then administered an intra-striatal IL-1β injection and two hours later plasma and brain were collected. IL-1β injection significantly increased striatal nSMase2 activity which was completely normalized by PDDC. Using SPRi, we found that IL-1β-induced injury selectively increased plasma levels of CD171 + and PLP1 + EVs; this EV increase was normalized by PDDC. In contrast, GLAST1 + EVs were unchanged by IL-1β or PDDC. IL-1β injection selectively increased EVs released from activated versus non-activated microglia, indicated by the CD11b+/IB4 + ratio. The increase in EVs from CD11b + microglia was dramatically attenuated with PDDC. Taken together, our data demonstrate that following acute injury, brain nSMase2 activity is elevated. EVs released from neurons, oligodendrocytes, and activated microglial are increased in plasma and inhibition of nSMase2 with PDDC reduced these IL-1β-induced changes implicating nSMase2 inhibition as a therapeutic target for acute brain injury.
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2022
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Quantitative proteomics identifies the core proteome of exosomes with syntenin-1 as the highest abundant protein and a putative universal biomarker
Exosomes are extracellular vesicles derived from the endosomal compartment that are potentially involved in intercellular communication. Here, we found that frequently used biomarkers of exosomes are heterogeneous, and do not exhibit universal utility across different cell types. To uncover ubiquitous and abundant proteins, we used an unbiased and quantitative proteomic approach based on super-stable isotope labeling with amino acids in cell culture (super-SILAC), coupled to high-resolution mass spectrometry. In total, 1,212 proteins were quantified in the proteome of exosomes, irrespective of the cellular source or isolation method. A cohort of 22 proteins was universally enriched. Fifteen proteins were consistently depleted in the proteome of exosomes compared to cells. Among the enriched proteins, we identified biogenesis-related proteins, GTPases and membrane proteins, such as CD47 and ITGB1. The cohort of depleted proteins in exosomes was predominantly composed of nuclear proteins. We identified syntenin-1 as a consistently abundant protein in exosomes from different cellular origins. Syntenin-1 is also present in exosomes across different species and biofluids, highlighting its potential use as a putative universal biomarker of exosomes. Our study provides a comprehensive quantitative atlas of core proteins ubiquitous to exosomes that can serve as a resource for the scientific community.
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2022
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Proteomic Profiling of Exosomes From Hemorrhagic Moyamoya Disease and Dysfunction of Mitochondria in Endothelial Cells
Background and Purpose: Moyamoya disease (MMD) is a rare steno-occlusive and slowly progressing cerebrovascular disorder. The detailed mechanism of the underlying pathogenesis is still blurry. Methods: Tandem Mass Tag-labeled quantitative proteomics was performed on serum-derived exosomes (SDEs) extracted from adult patients diagnosed with pure ischemic MMD or hemorrhagic MMD and healthy controls. Then mouse brain vascular endothelial cell (EC), human umbilical vein EC, neuroblastoma cell, and human hepatocyte cell were treated with exosomes, and changes of the protein expression in mouse brain vascular EC cells were identified. Results: Proteomics analysis results showed that 859 shared proteins were detected in SDEs from ischemic and hemorrhagic MMD patients with 231 differently expressed compared with healthy controls. Bioinformatic analysis revealed dysregulated cell growth and maintenance and indicated disturbed actin dynamics in MMD, with CFL1 (Cofilin-1) and ACTR2/3 (actin-related protein 2/3; also known as ARP2/3) downregulated in ischemic and hemorrhagic patients’ SDEs. We also found immunity dysfunction in hemorrhagic MMD. Following treatment with MMD SDEs, mouse brain vascular EC cells showed significantly higher levels of proliferation and more ethynyl-2-deoxyuridine-positive cells compared with the healthy control group, while there were no obvious changes in the human umbilical vein EC and human hepatocyte cell. Interestingly, we also found that SDEs from ischemic MMD promoted neuroblastoma cell proliferation. Proteomic analysis of mouse brain vascular EC cells suggested that SDEs from hemorrhagic MMD patients induced dysfunction of the mitochondria in cerebrovascular ECs. Conclusions: This study highlighted potential molecular mechanisms underlying the pathogenesis of MMD patients, thereby providing new therapeutic strategies for MMD.
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2022
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Modulation of Rumen Microbes Through Extracellular Vesicle Released by the Rumen Fluke Calicophoron daubneyi
Parasite derived extracellular vesicles (EVs) have been proposed to play key roles in the establishment and maintenance of infection. Calicophoron daubneyi is a newly emerging parasite of livestock with many aspects of its underpinning biology yet to be resolved. This research is the first in-depth investigation of EVs released by adult C. daubneyi. EVs were successfully isolated using both differential centrifugation and size exclusion chromatography (SEC), and morphologically characterized though transmission electron microscopy (TEM). EV protein components were characterized using a GeLC approach allowing the elucidation of comprehensive proteomic profiles for both their soluble protein cargo and surface membrane bound proteins yielding a total of 378 soluble proteins identified. Notably, EVs contained Sigma-class GST and cathepsin L and B proteases, which have previously been described in immune modulation and successful establishment of parasitic flatworm infections. SEC purified C. daubneyi EVs were observed to modulate rumen bacterial populations by likely increasing microbial species diversity via antimicrobial activity. This data indicates EVs released from adult C. daubneyi have a role in establishment within the rumen through the regulation of microbial populations offering new routes to control rumen fluke infection and to develop molecular strategies to improve rumen efficiency.
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2022
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Extracellular Vesicle Release Promotes Viral Replication during Persistent HCV Infection
Hepatitis C virus (HCV) infection promotes autophagic degradation of viral replicative intermediates for sustaining replication and spread. The excessive activation of autophagy can induce cell death and terminate infection without proper regulation. A prior publication from this laboratory showed that an adaptive cellular response to HCV microbial stress inhibits autophagy through beclin 1 degradation. The mechanisms of how secretory and degradative autophagy are regulated during persistent HCV infection is unknown. This study was performed to understand the mechanisms of viral persistence in the absence of degradative autophagy, which is essential for virus survival. Using HCV infection of a CD63-green fluorescence protein (CD63-GFP), labeled stable transfected Huh-7.5 cell, we found that autophagy induction at the early stage of HCV infection increased the degradation of CD63-GFP that favored virus replication. However, the late-stage of persistent HCV infection showed impaired autophagic degradation, leading to the accumulation of CD63-GFP. We found that impaired autophagic degradation promoted the release of extracellular vesicles and exosomes. The impact of blocking the release of extracellular vesicles (EVs) on virus survival was investigated in persistently infected cells and sub-genomic replicon cells. Our study illustrates that blocking EV and exosome release severely suppresses virus replication without effecting host cell viability. Furthermore, we found that blocking EV release triggers interferon lambda 1 secretion. These findings suggest that the release of EVs is an innate immune escape mechanism that promotes persistent HCV infection. We propose that inhibition of extracellular vesicle release can be explored as a potential antiviral strategy for the treatment of HCV and other emerging RNA viruses.
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2022
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Small Extracellular Vesicle Enrichment of a Retrotransposon-Derived Double-Stranded RNA: A Means to Avoid Autoinflammation?
Small extracellular vesicles (SEVs) such as exosomes are released by multiple cell types. Originally believed to be a mechanism for selectively removing unwanted cellular components, SEVs have received increased attention in recent years for their ability to mediate intercellular communication. Apart from proteins and lipids, SEVs contain RNAs, but how RNAs are selectively loaded into SEVs remains poorly understood. To address this question, we profiled SEV RNAs from mouse dendritic cells using RNA-Seq and identified a long noncoding RNA of retroviral origin, VL30, which is highly enriched (>200-fold) in SEVs compared to parental cells. Bioinformatic analysis revealed that exosome-enriched isoforms of VL30 RNA contain a repetitive 26-nucleotide motif. This repeated motif is itself efficiently incorporated into SEVs, suggesting the likelihood that it directly promotes SEV loading. RNA folding analyses indicate that the motif is likely to form a long double-stranded RNA hairpin and, consistent with this, its overexpression was associated with induction of a potent type I interferon response. Taken together, we propose that preferential loading into SEVs of the VL30 RNA containing this immunostimulatory motif enables cells to remove a potentially toxic RNA and avoid autoinflammation. In this way, the original notion of SEVs as a cellular garbage bin should not be entirely discounted.
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2022
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Stromal Cells Serve Drug Resistance for Multiple Myeloma via Mitochondrial Transfer: A Study on Primary Myeloma and Stromal Cells
Recently, it has become evident that mitochondrial transfer (MT) plays a crucial role in the acquisition of cancer drug resistance in many hematologic malignancies; however, for multiple myeloma, there is a need to generate novel data to better understand this mechanism. Here, we show that primary myeloma cells (MMs) respond to an increasing concentration of chemotherapeutic drugs with an increase in the acquisition of mitochondria from autologous bone marrow stromal cells (BM-MSCs), whereupon survival and adenosine triphosphate levels of MMs increase, while the mitochondrial superoxide levels decrease in MMs. These changes are proportional to the amount of incorporated BM-MSC-derived mitochondria and to the concentration of the used drug, but seem independent from the type and mechanism of action of chemotherapeutics. In parallel, BM-MSCs also incorporate an increasing amount of MM cell-derived mitochondria accompanied by an elevation of superoxide levels. Using the therapeutic antibodies Daratumumab, Isatuximab, or Elotuzumab, no similar effect was observed regarding the MT. Our research shows that MT occurs via tunneling nanotubes and partial cell fusion with extreme increases under the influence of chemotherapeutic drugs, but its inhibition is limited. However, the supportive effect of stromal cells can be effectively avoided by influencing the metabolism of myeloma cells with the concomitant use of chemotherapeutic agents and an inhibitor of oxidative phosphorylation.
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2022
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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-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.
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2022
Resveratrol loaded polymeric micelles for theranostic targeting of breast cancer cells
Treatment of breast cancer underwent extensive progress in recent years with molecularly targeted therapies. However, non-specific pharmaceutical approaches (chemotherapy) persist, inducing severe side-effects. Phytochemicals provide a promising alternative for breast cancer prevention and treatment. Specifically, resveratrol (res) is a plant-derived polyphenolic phytoalexin with potent biological activity but displays poor water solubility, limiting its clinical use. Here we have developed a strategy for delivering res using a newly synthesized nano-carrier with the potential for both diagnosis and treatment. Methods: Res-loaded nanoparticles were synthesized by the emulsion method using Pluronic F127 block copolymer and Vitamin E-TPGS. Nanoparticle characterization was performed by SEM and tunable resistive pulse sensing. Encapsulation Efficiency (EE%) and Drug Loading (DL%) content were determined by analysis of the supernatant during synthesis. Nanoparticle uptake kinetics in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in MCF-10A breast epithelial cells were evaluated by flow cytometry and the effects of res on cell viability via MTT assay. Results: Res-loaded nanoparticles with spherical shape and a dominant size of 179±22 nm were produced. Res was loaded with high EE of 73±0.9% and DL content of 6.2±0.1%. Flow cytometry revealed higher uptake efficiency in breast cancer cells compared to the control. An MTT assay showed that res-loaded nanoparticles reduced the viability of breast cancer cells with no effect on the control cells. Conclusions: These results demonstrate that the newly synthesized nanoparticle is a good model for the encapsulation of hydrophobic drugs. Additionally, the nanoparticle delivers a natural compound and is highly effective and selective against breast cancer cells rendering this type of nanoparticle an excellent candidate for diagnosis and therapy of difficult to treat mammary malignancies.
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2022
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Exosomes from neuronal stem cells may protect the heart from ischaemia/reperfusion injury via JAK1/2 and gp130
Myocardial infarction requires urgent reperfusion to salvage viable heart tissue. However, reperfusion increases infarct size further by promoting mitochondrial damage in cardiomyocytes. Exosomes from a wide range of different cell sources have been shown to activate cardioprotective pathways in cardiomyocytes, thereby reducing infarct size. Yet, it is currently challenging to obtain highly pure exosomes in quantities enough for clinical studies. To overcome this problem, we used exosomes isolated from CTX0E03 neuronal stem cells, which are genetically stable, conditionally inducible and can be produced on an industrial scale. However, it is unknown whether exosomes from neuronal stem cells may reduce cardiac ischaemia/reperfusion injury. In this study, we demonstrate that exosomes from differentiating CTX0E03 cells can reduce infarct size in mice. In an in vitro assay, these exosomes delayed cardiomyocyte mitochondrial permeability transition pore opening, which is responsible for cardiomyocyte death after reperfusion. The mechanism of MPTP inhibition was via gp130 signalling and the downstream JAK/STAT pathway. Our results support previous findings that exosomes from non-cardiomyocyte-related cells produce exosomes capable of protecting cardiomyocytes from myocardial infarction. We anticipate our findings may encourage scientists to use exosomes obtained from reproducible clinical-grade stocks of cells for their ischaemia/reperfusion studies.
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2022
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Breast cancer is the leading cause of cancer death in women. The majority of these deaths are due to disease metastasis, in which cancer cells disseminate to multiple organs and disrupt vital physiological functions. It is widely accepted that breast cancer cells secrete extracellular vesicles (EVs), which contain dynamic molecular cargo that act as versatile mediators of intercellular communication. Therefore, Evs. secreted by breast cancer cells could be involved in the development of metastatic disease and resistance to treatment. Moreover, changes in EV cargo could reflect the effects of therapy on their parent tumor cells. The aim of this feasibility study was to quantitatively profile the proteomes of Evs. isolated from blood samples taken from treatment sensitive and resistant metastatic breast cancer patients to identify proteins associated with responses. Three serial blood samples were collected from three patients with metastatic breast cancer receiving systemic therapy including a responder, a non-responder, and a mixed-responder. Evs. were isolated from plasma using size exclusion chromatography and their protein cargo was prepared for tandem mass tag (TMT)-labelling and quantitative analyses using two-dimensional high-performance liquid chromatography followed by tandem mass spectrometry. After filtering, we quantitatively identified 286 proteins with high confidence using a q value of 0.05. Of these, 149 were classified as EV associated candidate proteins and 137 as classical, high abundant plasma proteins. After comparing EV protein abundance between the responder and non-responder, we identified 35 proteins with unique de-regulated abundance patterns that was conserved at multiple time points. We propose that this proof-of-concept approach can be used to identify proteins which have potential as predictors of metastatic breast cancer response to treatment.
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2022
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A novel strategy to identify candidate diagnostic and prognostic biomarkers for gastric cancer
BackgroundGastric cancer (GC) is one of the most common cancer worldwide. It is essential to identify non-invasive diagnostic and prognostic biomarkers of GC. The aim of the present study was to screen candidate biomarkers associated with the pathogenesis and prognosis of GC by a novel strategy.MethodsThe expression level of gene higher in cancer than in adjacent non-cancer tissue was defined as “positive”, and the top 5% genes with “positive rate” were filtered out as candidate diagnostic biomarkers in three Gene Expression Omnibus (GEO) datasets. Further, a prognostic risk model was constructed by multivariate Cox regression analysis in GEO dataset and validated in The Cancer Genome Atlas (TCGA). The expression level of candidate biomarkers was determined in serum and serum-derived exosomes of GC patients. Moreover, the effect of biomarkers in exosomes on migration of GC cells was analyzed by transwell assay.ResultsTen candidate biomarkers (AGT, SERPINH1, WNT2, LIPG, PLAU, COL1A1, MMP7, MXRA5, CXCL1 and COL11A1) were identified with efficient diagnostic value in GC. A prognostic gene signature consisted of AGT, SERPINH1 and MMP7 was constructed and showed a good performance in predicting overall survivals in TCGA. Consistently, serum levels of the three biomarkers also showed high sensitivity and specificity in distinguishing GC patients from controls. In addition, the expression level of the three biomarkers were associated with malignant degree and decreased after surgery in GC patients. Moreover, the expression level of AGT and MMP7 in exosomes correlated positively with serum level. The exosomes derived from serum of GC patients can promote migration of SGC‐7901 cells. After neutralized the expression level of three proteins in exosomes with antibodies, the migration of GC cells was obviously suppressed.ConclusionsOur findings provided a novel strategy to identify diagnostic biomarkers based on public datasets, and suggested that the three-gene signature was a candidate diagnostic and prognostic biomarker for patients with GC.
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2022
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Differentially Expressed Extracellular Vesicle, Exosome and Non-Exosome miRNA Profile in High and Low Tick-Resistant Beef Cattle
Heavy tick burden on beef cattle account for huge economic losses globally, with an estimated value of US$22-30 billion per annum. In Australia, ticks cost the northern beef industry approximately A$170-200 million. Methods to evaluate and predict tick resistance would therefore be of great value to the global cattle trade. Exosomes (EX) are small extracellular vesicles (EVs) of ~30-150nm diameter and have gained popularity for their diagnostic and prognostic potential. EX contain, among other biomolecules, various types of RNA including micro-RNA (miRNA) and long noncoding RNA (lncRNA). MiRNA specifically have been validated as therapeutic biomarkers as they perform regulatory functions at the post-transcriptional level and are differentially expressed between divergent groups. The objective of the present study was to evaluate the miRNA profiles of EV and fractionated exosomal samples of high and low tick-resistant beef cattle to highlight potential miRNA biomarkers of tick resistance. Cows (n = 3/group) were classified into high or low tick resistant groups according to a novel scoring system. EVs and EX were isolated and fractionated from the blood plasma of high and low tick resistant cattle using established isolation and enrichment protocols. The resultant EX and non-EX samples were processed for next generation miRNA sequencing. Offspring of the cows in each high and low tick resistant group underwent the same processing for blood plasma EX, non-EX and miRNA analysis to evaluate the heritability of miRNA associated with tick resistance. A total of 2631 miRNAs were identified in EX and non-EX fractionated samples from high and low tick-resistant beef cattle. MiR-449a was highly expressed in maternal high tick-resistant EX samples. Of these, 174 were novel miRNAs, and 10 were differentially expressed (DE) (FDR < 0.05). These 10 DE miRNAs were also present in EVs, and three miRNAs were highly expressed: miR-2419-3p, miR-7861-3p and miR-2372-5p. Although 196 novel miRNAs were identified in fractionated samples of offspring, no miRNA were differentially expressed in these animals.
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2022
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Evidence of Immune Modulators in the Secretome of the Equine Tapeworm Anoplocephala perfoliata
Anoplocephala perfoliata is a neglected gastro-intestinal tapeworm, commonly infecting horses worldwide. Molecular investigation of A. perfoliata is hampered by a lack of tools to better understand the host-parasite interface. This interface is likely influenced by parasite derived immune modulators released in the secretome as free proteins or components of extracellular vesicles (EVs). Therefore, adult RNA was sequenced and de novo assembled to generate the first A. perfoliata transcriptome. In addition, excretory secretory products (ESP) from adult A. perfoliata were collected and EVs isolated using size exclusion chromatography, prior to proteomic analysis of the EVs, the EV surface and EV depleted ESP. Transcriptome analysis revealed 454 sequences homologous to known helminth immune modulators including two novel Sigma class GSTs, five α-HSP90s, and three α-enolases with isoforms of all three observed within the proteomic analysis of the secretome. Furthermore, secretome proteomics identified common helminth proteins across each sample with known EV markers, such as annexins and tetraspanins, observed in EV fractions. Importantly, 49 of the 454 putative immune modulators were identified across the secretome proteomics contained within and on the surface of EVs in addition to those identified in free ESP. This work provides the molecular tools for A. perfoliata to reveal key players in the host-parasite interaction within the horse host.
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2022
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Unique somatic variants in DNA from urine exosomes of individuals with bladder cancer
Bladder cancer (BC), a heterogeneous disease characterized by high recurrence rates, is diagnosed and monitored by cystoscopy. Accurate clinical staging based on biopsy remains a challenge, and additional, objective diagnostic tools are needed urgently. We used exosomal DNA (exoDNA) as an analyte to examine cancer-associated mutations and compared the diagnostic utility of exoDNA from urine and serum of individuals with BC. In contrast to urine exosomes from healthy individuals, urine exosomes from individuals with BC contained significant amounts of DNA. Whole-exome sequencing of DNA from matched urine and serum exosomes, bladder tumors, and normal tissue (peripheral blood mononuclear cells) identified exonic and 3' UTR variants in frequently mutated genes in BC, detectable in urine exoDNA and matched tumor samples. Further analyses identified somatic variants in driver genes, unique to urine exoDNA, possibly because of the inherent intra-tumoral heterogeneity of BC, which is not fully represented in random small biopsies. Multiple variants were also found in untranslated portions of the genome, such as microRNA (miRNA)-binding regions of the KRAS gene. Gene network analyses revealed that exoDNA is associated with cancer, inflammation, and immunity in BC exosomes. Our findings show utility of exoDNA as an objective, non-invasive strategy to identify novel biomarkers and targets for BC.
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2022
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Proteomic Profiling of Extracellular Vesicles Separated from Plasma of Former National Football League Players at Risk for Chronic Traumatic Encephalopathy
Chronic Traumatic Encephalopathy (CTE) is a tauopathy that affects individuals with a history of exposure to repetitive head impacts, including National Football League (NFL) players. Extracellular vesicles (EVs) are known to carry tau in Alzheimer's disease and other tauopathies. We examined protein profiles of EVs separated from the plasma of former NFL players at risk for CTE. EVs were separated from the plasma from former NFL players and age-matched controls using size-exclusion chromatography. Label-free quantitative proteomic analysis identified 675 proteins in plasma EVs, and 17 proteins were significantly differentially expressed between former NFL players and controls. Total tau (t-tau) and tau phosphorylated at threonie181 (p-tau181) in plasma-derived EVs were measured by ultrasensitive immunoassay. Level of t-tau and p-tau181 in EVs were significantly different, and the area under the receiver operating characteristic curve (AUC) of t-tau and p-tau181 showed 0.736 and 0.715, respectively. Machine learning analysis indicated that a combination of collagen type VI alpha 3 and 1 chain (COL6A3 and COL6A1) and reelin (RELN) can distinguish former NFL players from controls with 85% accuracy (AUC = 0.85). Based on the plasma EV proteomics, these data provide protein profiling of plasma EVs for CTE, and indicate combination of COL6A3, RELN and COL6A1 in plasma EVs may serve as the potential diagnostic biomarkers for CTE.
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2022
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Multiplexed electrokinetic sensor for detection and therapy monitoring of extracellular vesicles from liquid biopsies of non-small-cell lung cancer patients
Liquid biopsies based on extracellular vesicles (EVs) represent a promising tool for treatment monitoring of tumors, including non-small-cell lung cancers (NSCLC). In this study, we report on a multiplexed electrokinetic sensor for surface protein profiling of EVs from clinical samples. The method detects the difference in the streaming current generated by EV binding to the surface of a functionalized microcapillary, thereby estimating the expression level of a marker. Using multiple microchannels functionalized with different antibodies in a parallel fluidic connection, we first demonstrate the capacity for simultaneous detection of multiple surface markers in small EVs (sEVs) from NSCLC cells. To investigate the prospects of liquid biopsies based on EVs, we then apply the method to profile sEVs isolated from the pleural effusion (PE) fluids of five NSCLC patients with different genomic alterations (ALK, KRAS or EGFR) and applied treatments (chemotherapy, EGFR- or ALK-tyrosine kinase inhibitors). The vesicles were targeted against CD9, as well as EGFR and PD-L1, two treatment targets in NSCLC. The electrokinetic signals show detection of these markers on sEVs, highlighting distinct interpatient differences, e.g., increased EGFR levels in sEVs from a patient with EGFR mutation as compared to an ALK-fusion one. The sensors also detect differences in PD-L1 expressions. The analysis of sEVs from a patient prior and post ALK-TKI crizotinib treatment reveals significant increases in the expressions of some markers (EGFR and PD-L1). These results hold promise for the application of the method for tumor treatment monitoring based on sEVs from patient liquid biopsies.
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2022
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Extracellular vesicle‐mediated endothelial apoptosis and EV‐associated proteins correlate with COVID‐19 disease severity
Coronavirus disease-2019 (COVID-19), caused by the novel severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has lead to a global pandemic with a rising toll in infections and deaths. Better understanding of its pathogenesis will greatly improve the outcomes and treatment of affected patients. Here we compared the inflammatory and cardiovascular disease-related protein cargo of circulating large and small extracellular vesicles (EVs) from 84 hospitalized patients infected with SARS-CoV-2 with different stages of disease severity. Our findings reveal significant enrichment of proinflammatory, procoagulation, immunoregulatory and tissue-remodelling protein signatures in EVs, which remarkably distinguished symptomatic COVID-19 patients from uninfected controls with matched comorbidities and delineated those with moderate disease from those who were critically ill. Specifically, EN-RAGE, followed by TF and IL-18R1, showed the strongest correlation with disease severity and length of hospitalization. Importantly, EVs from COVID-19 patients induced apoptosis of pulmonary microvascular endothelial cells in the order of disease severity. In conclusion, our findings support a role for EVs in the pathogenesis of COVID-19 disease and underpin the development of EV-based approaches to predicting disease severity, determining need for patient hospitalization and identifying new therapeutic targets.
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2022
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Molecular evaluation of five different isolation methods for extracellular vesicles reveals different clinical applicability and subcellular origin
Extracellular vesicles (EVs) are increasingly tested as therapeutic vehicles and biomarkers, but still EV subtypes are not fully characterised. To isolate EVs with few co-isolated entities, a combination of methods is needed. However, this is time-consuming and requires large sample volumes, often not feasible in most clinical studies or in studies where small sample volumes are available. Therefore, we compared EVs rendered by five commonly used methods based on different principles from conditioned cell medium and 250 μl or 3 ml plasma, that is, precipitation (ExoQuick ULTRA), membrane affinity (exoEasy Maxi Kit), size-exclusion chromatography (qEVoriginal), iodixanol gradient (OptiPrep), and phosphatidylserine affinity (MagCapture). EVs were characterised by electron microscopy, Nanoparticle Tracking Analysis, Bioanalyzer, flow cytometry, and LC-MS/MS. The different methods yielded samples of different morphology, particle size, and proteomic profile. For the conditioned medium, Izon 35 isolated the highest number of EV proteins followed by exoEasy, which also isolated fewer non-EV proteins. For the plasma samples, exoEasy isolated a high number of EV proteins and few non-EV proteins, while Izon 70 isolated the most EV proteins. We conclude that no method is perfect for all studies, rather, different methods are suited depending on sample type and interest in EV subtype, in addition to sample volume and budget.
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2022
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Label-Free Classification of Bacterial Extracellular Vesicles by Combining Nanoplasmonic Sensors With Machine Learning
Bacterial extracellular vesicles (EVs) are nano- scale lipid-enclosed packages that are released by bacteria cells and shuttle various biomolecules between bacteria or host cells. They are implicated in playing several important roles, from infectious disease progression to maintaining proper gut health, however the tools available to characterise and classify them are limited and impractical for many applications. Surface-enhanced Raman Spectroscopy (SERS) provides a promising means of rapidly fingerprinting bacterial EVs in a label-free manner by taking advantage of plasmonic resonances that occur on nanopatterned surfaces, effectively amplifying the inelastic scattering of incident light. In this study, we demonstrate that by applying machine learning algorithms to bacterial EV SERS spectra, EVs from cultures of the same bacterial species (Escherichia coli) can be classified by strain, culture conditions, and purification method. While these EVs are highly purified and homogeneous compared to complex samples, the ability to classify them from a single species demonstrates the incredible power of SERS when combined with machine learning, and the importance of considering these parameters in future applications. We anticipate that these findings will play a crucial role in developing the laboratory and clinical utility of bacterial EVs, such as the label-free, noninvasive, and rapid diagnosis of infections without the need to culture samples from blood, urine, or other fluids.
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2022
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Developmental Timing of Trauma in Women Predicts Unique Extracellular Vesicle Proteome Signatures
BACKGROUND: Exposure to traumatic events is a risk factor for negative physical and mental health outcomes. However, the underlying biological mechanisms that perpetuate these lasting effects are not known. METHODS: We investigated the impact and timing of sexual trauma, a specific type of interpersonal violence, experienced during key developmental windows of childhood, adolescence, or adulthood on adult health outcomes and associated biomarkers, including circulating cell-free mitochondrial DNA levels and extracellular vesicles (EVs), in a predominantly Black cohort of women (N = 101). RESULTS: Significant changes in both biomarkers examined, circulating cell-free mitochondrial DNA levels and EV proteome, were specific to developmental timing of sexual trauma. Specifically, we identified a large number of keratin-related proteins from EVs unique to the adolescent sexual trauma group. Remarkably, the majority of these keratin proteins belong to a 17q21 gene cluster, which suggests a potential local epigenetic regulatory mechanism altered by adolescent trauma to impact keratinocyte EV secretion or its protein cargo. These results, along with changes in fear-potentiated startle and skin conductance detected in these women, suggest that sexual violence experienced during the specific developmental window of adolescence may involve unique programming of the skin, the body's largest stress organ. CONCLUSIONS: Together, these descriptive studies provide novel insight into distinct biological processes altered by trauma experienced during specific developmental windows. Future studies will be required to mechanistically link these biological processes to health outcomes.
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2022
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Extracellular vesicles are the primary source of blood‐borne tumour‐derived mutant KRAS DNA early in pancreatic cancer
Up to now, the field of liquid biopsies has focused on circulating tumour DNA and cells, though extracellular vesicles (EVs) have been of increasing interest in recent years. Thus, reported sources of tumour-derived nucleic acids include leukocytes, platelets and apoptotic bodies (AB), as well as large (LEV) and small (SEV) EVs. Despite these competing claims, there has yet to be a standardized comparison of the tumour-derived DNA associated with different components of blood. To address this issue, we collected twenty-three blood samples from seventeen patients with pancreatic cancers of known mutant KRAS G12 genotype, and divided them into two groups based on the time of patient survival following sampling. After collecting red and white blood cells, we subjected 1 ml aliquots of platelet rich plasma to differential centrifugation in order to separate the platelets, ABs, LEVs, SEVs and soluble proteins (SP) present. We then confirmed the enrichment of specific blood components in each differential centrifugation fraction using electron microscopy, Western blotting, nanoparticle tracking analysis and bead-based multiplex flow cytometry assays. By targeting wild type and tumour-specific mutant KRAS alleles using digital PCR, we found that the levels of mutant KRAS DNA were highest in association with LEVs and SEVs early, and with SEVs and SP late in disease progression. Importantly, we established that SEVs were the most enriched in tumour-derived DNA throughout disease progression, and verified this association using size exclusion chromatography. This work provides important direction for the rapidly expanding field of liquid biopsies by supporting an increased focus on EVs as a source of tumour-derived DNA.
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2022
α‐Synuclein in Plasma‐Derived Extracellular Vesicles Is a Potential Biomarker of Parkinson's Disease
BACKGROUND: Extracellular vesicles are small vesicles that are released from many cells, including neurons. α-Synuclein has recently been described in extracellular vesicles derived from the central nervous system and may contribute to the spreading of disease pathology in α-synuclein-related neurodegeneration. OBJECTIVES: We aimed to examine the potential diagnostic value of α-synuclein in plasma extracellular vesicles from patients with Parkinson's disease (PD). METHODS: Preanalytical variables were studied to establish an optimized assay for preparation of plasma extracellular vesicles and detection of extracellular vesicle-derived α-synuclein. Plasma samples were obtained from 2 independent cohorts. The Tübingen cohort contained 96 patients with PD, 50 patients with dementia with Lewy bodies, 50 patients with progressive supranuclear palsy (PSP), and 42 healthy controls; the Kassel cohort included 47 patients with PD, 43 patients with dementia with Lewy bodies, and 36 controls with secondary parkinsonian syndromes. Extracellular vesicles were prepared from total plasma by size exclusion chromatography and quantified by nanoparticle tracking analysis, α-synuclein content was measured by an electrochemiluminescence assay. RESULTS: α-Synuclein concentration in plasma extracellular vesicles provided the best discrimination between PD, dementia with Lewy bodies, PSP, and healthy controls, with an area under the curve of 0.804 (PD vs dementia with Lewy bodies), 0.815 (PD vs. PSP), and 0.769 (PD vs healthy controls) in the Tübingen cohort. Results were validated in the Kassel cohort. CONCLUSIONS: The concentration of α-synuclein in plasma extracellular vesicles may serve as a potential diagnostic biomarker for PD. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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2022
nm
T908 Polymeric Micelles Improved the Uptake of Sgc8-c Aptamer Probe in Tumor-Bearing Mice: A Co-Association Study between the Probe and Preformed Nanostructures
Aptamers are oligonucleotides that have the characteristic of recognizing a target with high affinity and specificity. Based on our previous studies, the aptamer probe Sgc8-c-Alexa647 is a promising tool for molecular imaging of PTK7, which is an interesting biomarker in cancer. In order to improve the delivery of this probe as well as create a novel drug delivery nanosystem targeted to the PTK7 receptor, we evaluate the co-association between the probe and preformed nanostructures. In this work, preformed pegylated liposomes (PPL) and linear and branched pristine polymeric micelles (PMs), based on PEO-PPO-PEO triblock copolymers were used: poloxamer F127® and poloxamines T1307® and T908®. For it, Sgc8-c-Alexa647 and its co-association with the different nanostructures was exhaustively analyzed. DLS analysis showed nanometric sizes, and TEM and AFM showed notable differences between free- and co-associated probe. Likewise, all nanosystems were evaluated on A20 lymphoma cell line overexpressing PTK7, and the confocal microscopy images showed distinctness in cellular uptake. Finally, the biodistribution in BALB/c mice bearing lymphoma-tumor and pharmacokinetic study revealed an encouraging profile for T908-probe. All data obtained from this work suggested that PMs and, more specifically T908 ones, are good candidates to improve the pharmacokinetics and the tumor uptake of aptamer-based probes.
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2022
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Isolation of intact extracellular vesicles from cryopreserved samples
Extracellular vesicles (EVs) have emerged as promising candidates in biomarker discovery and diagnostics. Protected by the lipid bilayer, the molecular content of EVs in diverse biofluids are protected from RNases and proteases in the surrounding environment that may rapidly degrade targets of interests. Nonetheless, cryopreservation of EV-containing samples to -80°C may expose the lipid bilayer to physical and biological stressors which may result in cryoinjury and contribute to changes in EV yield, function, or molecular cargo. In the present work, we systematically evaluate the effect of cryopreservation at -80°C for a relatively short duration of storage (up to 12 days) on plasma- and media-derived EV particle count and/or RNA yield/quality, as compared to paired fresh controls. On average, we found that the plasma-derived EV concentration of stored samples decreased to 23% of fresh samples. Further, this significant decrease in EV particle count was matched with a corresponding significant decrease in RNA yield whereby plasma-derived stored samples contained only 47-52% of the total RNA from fresh samples, depending on the extraction method used. Similarly, media-derived EVs showed a statistically significant decrease in RNA yield whereby stored samples were 58% of the total RNA from fresh samples. In contrast, we did not obtain clear evidence of decreased RNA quality through analysis of RNA traces. These results suggest that samples stored for up to 12 days can indeed produce high-quality RNA; however, we note that when directly comparing fresh versus cryopreserved samples without cryoprotective agents there are significant losses in total RNA. Finally, we demonstrate that the addition of the commonly used cryoprotectant agent, DMSO, alongside greater control of the rate of cooling/warming, can rescue EVs from damaging ice formation and improve RNA yield.
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2022
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Choice of selectable marker affects recombinant protein expression in cells and exosomes
Transgenic mammalian cells are used for numerous research, pharmaceutical, industrial, and clinical purposes, and dominant selectable markers are often used to enable the selection of transgenic cell lines. Using HEK293 cells, we show here that the choice of selectable marker gene has a significant impact on both the level of recombinant protein expression and the cell-to-cell variability in recombinant protein expression. Specifically, we observed that cell lines generated with the NeoR or BsdR selectable markers and selected in the antibiotics G418 or blasticidin, respectively, displayed the lowest level of recombinant protein expression as well as the greatest cell-to-cell variability in transgene expression. In contrast, cell lines generated with the BleoR marker and selected in zeocin yielded cell lines that expressed the highest levels of linked recombinant protein, approximately 10-fold higher than those selected using the NeoR or BsdR markers, as well as the lowest cell-to-cell variability in recombinant protein expression. Intermediate yet still-high levels of expression were observed in cells generated with the PuroR- or HygR-based vectors and that were selected in puromycin or hygromycin, respectively. Similar results were observed in the African green monkey cell line COS7. These data indicate that each combination of selectable marker and antibiotic establishes a threshold below which no cell can survive and that these thresholds vary significantly between different selectable markers. Moreover, we show that choice of selectable marker also affects recombinant protein expression in cell-derived exosomes, consistent with the hypothesis that exosome protein budding is a stochastic rather than determinative process.
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2022
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Exosomes derived from bone marrow mesenchymal stromal cells promote remyelination and reduce neuroinflammation in the demyelinating central nervous system
Injury of oligodendrocytes (OLs) induces demyelination, and patients with neurodegenerative diseases exhibit demyelination concomitantly with neurological deficit and cognitive impairment. Oligodendrocyte progenitor cells (OPCs) are present in the adult central nervous system (CNS), and they can proliferate, differentiate, and remyelinate axons after damage. However, remyelination therapies are not in clinical use. Multiple sclerosis (MS) is a major demyelinating disease in the CNS. Mesenchymal stromal cells (MSCs) have demonstrated therapeutic promise in animal models and in clinical trials of MS. Exosomes are nanoparticles generated by nearly all cells and they mediate cell-cell communication by transferring cargo biomaterials. Here, we hypothesize that exosomes harvested from MSCs have a similar therapeutic effect on enhancement of remyelination as that of MSCs. In the present study we employed exosomes derived from rhesus monkey MSCs (MSC-Exo). Two mouse models of demyelination were employed: 1) experimental autoimmune encephalomyelitis (EAE), an animal model of MS; and 2) cuprizone (CPZ) diet model, a toxic demyelination model. MSC-Exo or PBS were intravenously injected twice a week for 4 weeks, starting on day 10 post immunization in EAE mice, or once a week for 2 weeks starting on the day of CPZ diet withdrawal. Neurological and cognitive function were tested, OPC differentiation and remyelination, neuroinflammation and the potential underlying mechanisms were investigated using immunofluorescent staining, transmission electron microscopy and Western blot. Data generated from the EAE model revealed that MSC-Exo cross the blood brain barrier (BBB) and target neural cells. Compared with the controls (p < 0.05), treatment with MSC-Exo: 1) significantly improved neurological outcome; 2) significantly increased the numbers of newly generated OLs (BrdU+/APC+) and mature OLs (APC+), and the level of myelin basic protein (MBP); 3) decreased amyloid-β precursor protein (APP)+ density; 4) decreased neuroinflammation by increasing the M2 phenotype and decreasing the M1 phenotype of microglia, as well as their related cytokines; 5) inhibited the TLR2/IRAK1/NFκB pathway. Furthermore, we confirmed that the MSC-Exo treatment significantly improved cognitive function, promoted remyelination, increased polarization of M2 phenotype and blocked TLR2 signaling in the CPZ model. Collectively, MSC-Exo treatment promotes remyelination by both directly acting on OPCs and indirectly by acting on microglia in the demyelinating CNS. This study provides the cellular and molecular basis for this cell-free exosome therapy on remyelination and modulation of neuroinflammation in the CNS, with great potential for treatment of demyelinating and neurodegenerative disorders.
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2022
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Human urine-derived stem cell-derived exosomal miR-21-5p promotes neurogenesis to attenuate Rett syndrome via the EPha4/TEK axis
Rett syndrome (RTT) is a rare neurodevelopmental disorder that results in multiple disabilities. Exosomal microRNA (miRs) from urine-derived stem cells (USCs) have been shown to induce neurogenesis and aid in functional recovery from brain ischemia. In the present study, we sought to determine whether that exosomal miR-21-5p from USCs could promote early neural formation in a model of RTT. USCs were isolated and evaluated by flow cytometry. Exosomes were analyzed by transmission electron microscopy, tunable resistive pulse sensing (TRPS), and western blotting. PKH26 fluorescent dyes were used to observe intake of exosomes in vivo and in vitro. An RTT mouse model was treated with exosomes for behavioral studies. Dual‐luciferase report gene assays were conducted to evaluate the relationship between miR-21-5p and Eph receptor A4 (EphA4). In vitro, treatment with exosomes from human urine‐derived stem cells (USC-Exos) increased the percentage of neuron-specific class III beta-tubulin (Tuj1)+ nerve cells as well as the transcription levels of β-III tubulin and doublecortin (DCX). A higher level of miR-21-5p was observed in USC-Exos, which promoted differentiation in NSCs by targeting the EPha4/TEK axis. In vivo, exosomal miR-21-5p improved the behavior, motor coordination, and cognitive ability of mice, facilitated the differentiation of NSCs in the subventricular zone of the lateral ventricle and promoted a marked rise in the number of DCX+ cells. Our data provide evidence that exosomal miR-21-5p from human USCs facilitate early nerve formation by regulating the EPha4/TEK axis.
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2022
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Telocytes‐derived extracellular vesicles alleviate aortic valve calcification by carrying miR‐30b
AIMS: Calcific aortic valve disease (CAVD) is frequent in the elderly. Telocytes (TCs) are implicated in intercellular communication by releasing extracellular vesicles (EVs). This study investigated the role of TC-EVs in aortic valve calcification. METHODS AND RESULTS: TCs were obtained and identified using enzymolysis method and flow cytometry. EVs were isolated from TCs using differential high-speed centrifugation method and identified using transmission electron microscope, western blot, and qNano analysis. The mouse model of CAVD was established. The changes of aortic valve activity-related indicators were analysed by ultrasound, and the expressions of TC markers CD34 and vimentin in mouse valve tissues were detected using RT-qPCR and western blot. The model mice were injected with TC-derived EVs. The expressions of Runx2, osteocalcin, and caspase-3 were detected using RT-qPCR and western blot. The calcification model of valvular interstitial cells (VICs) was established. TC-EVs were co-cultured with calcified VICs, and calcium deposition was detected using alizarin red S staining. miR-30b expression in calcified valvular tissues and cells was detected after EV treatment. miR-30b expression in TCs was knocked down and then EVs were extracted and co-cultured with calcified VICs. The target of miR-30b was predicted through bioinformatics website and verified using dual-luciferase assay. The levels of Wnt/β-catenin pathway-related proteins were detected. ApoE-/- mice fed with a high-fat diet showed decreased aortic valve orifice area, increased aortic transvalvular pressure difference and velocity, reduced left ventricular ejection fraction, decreased CD34 and vimentin, and increased caspase-3, Runx2, and osteocalcin. The levels of apoptosis- and osteogenesis- related proteins were inhibited after EV treatment. TC-EVs reduced calcium deposition and osteogenic proteins in calcified VICs. EVs could be absorbed by VICs. miR-30b expression was promoted in calcified valvular tissues and cells after EV treatment. Knockdown of miR-30b weakened the inhibitory effects of TC-EVs on calcium deposition and osteogenic proteins. miR-30b targeted Runx2. EV treatment inhibited the Wnt/β-catenin pathway, and knockdown of miR-30b in TCs attenuated the inhibitory effect of TC-EVs on the Wnt/β-catenin pathway. CONCLUSION: TC-EVs played a protective role in aortic valve calcification via the miR-30b/Runx2/Wnt/β-catenin axis.
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2022
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Influence of Anti-Glaucoma Drugs on Uptake of Extracellular Vesicles by Trabecular Meshwork Cells
BACKGROUND: Extracellular vesicles (EVs) are capable of manipulating cellular functions for the maintenance of biological homeostasis and disease progression, such as in glaucoma disease. These nano-particles carry a net negative surface charge under physiological conditions that can contribute to EVs:EVs interaction and their uptake by target cells. PURPOSE: To investigate the effect of glaucoma drugs on EVs physicochemical characters and the implications for their uptake by trabecular meshwork (TM) cells. METHODS: TM or non-pigmented ciliary epithelium (NPCE) cells derived EVs were incubated with commercial anti-glaucoma formulation, Timolol maleate, Brinzolamide or Benzalkonium Cl and their size and zeta potential (ZP) and physical interactions of EVs derived from NPCE cells and TM cells were evaluated. The contribution of EVs interactions to up-take by TM cells was examined using fluorescence-activated cell sorting. RESULTS: EVs size and ZP were affected by the ionic strength of the buffer rather than EVs type. Commercial glaucoma eye drops, including β-blocker, α-2-agonist and prostaglandin analogs, reduced NPCE EVs ZP, whereas exposure of EVs to carbonic anhydrase inhibitor caused an increase in the ZP. A correlation was found between increased ZP values and increased NPCE EVs uptake by TM cells. We were able to show that Benzalkonium chloride stands behind this ZP effect and not Timolol or Brinzolamide. CONCLUSION: Altogether, our findings demonstrate that EVs size, surface membrane charge, and ionic strength of the surrounding have an impact on EVs:EVs interactions, which affect the uptake of NPCE EVs by TM cells.
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2022
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Role of peroxiredoxin 6 in the chondroprotective effects of microvesicles from human adipose tissue-derived mesenchymal stem cells
BACKGROUND: Osteoarthritis (OA) is a joint disease characterized by cartilage degradation, low-grade synovitis and subchondral bone alterations. In the damaged joint, there is a progressive increase of oxidative stress leading to disruption of chondrocyte homeostasis. The modulation of oxidative stress could control the expression of inflammatory and catabolic mediators involved in OA. We have previously demonstrated that extracellular vesicles (EVs) present in the secretome of human mesenchymal stem cells from adipose tissue (AD-MSCs) exert anti-inflammatory and anti-catabolic effects in OA chondrocytes. In the current work, we have investigated whether AD-MSC EVs could regulate oxidative stress in OA chondrocytes as well as the possible contribution of peroxiredoxin 6 (Prdx6). METHODS: Microvesicles (MV) and exosomes (EX) were isolated from AD-MSC conditioned medium by differential centrifugation with size filtration. The size and concentration of EVs were determined by resistive pulse sensing. OA chondrocytes were isolated from knee articular cartilage of advanced OA patients. 4-Hydroxynonenal adducts, IL-6 and MMP-13 were determined by enzyme-linked immunosorbent assay. Expression of Prdx6 and autophagic markers was assessed by immunofluorescence and Western blotting. Prdx6 was downregulated in AD-MSCs by transfection with a specific siRNA. RESULTS: MV and to a lesser extent EX significantly reduced the production of oxidative stress in OA chondrocytes stimulated with IL-1β. Treatment with MV resulted in a dramatic upregulation of Prdx6. MV also enhanced the expression of autophagy marker LC3B. We downregulated Prdx6 in AD-MSCs by using a specific siRNA and then MV were isolated. These Prdx6-silenced MV failed to modify oxidative stress and the expression of autophagy markers. We also assessed the possible contribution of Prdx6 to the effects of MV on IL-6 and MMP-13 production. The reduction in the levels of both mediators induced by MV was partly reverted after Prdx6 silencing. CONCLUSION: Our results indicate that EVs from AD-MSCs regulate the production of oxidative stress in OA chondrocytes during inflammation. Prdx6 may mediate the antioxidant and protective effects of MV.The translational potential of this article: This study gives insight into the protective properties of EVs from AD-MSCs in OA chondrocytes. Our findings support the development of novel therapies based on EVs to prevent or treat cartilage degradation.
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2022
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An increasing number of individuals are suffering from lower back and neck pain caused by intervertebral disc degeneration each year. Although the application of mesenchymal stem cells (MSCs) has provided desirable results in the treatment of intervertebral disc degeneration, there are multiple risks associated with the directed application of MSCs. An increasing number of studies have suggested that stem cells, through the release of extracellular nanovesicles, have vital functions in tissue regeneration and repair with low risk. The present study investigated the effect of extracellular nanovesicles derived from adipose-derived stem cells (ADSCs) on nucleus pulposus (NP) cells from patients with intervertebral disc degeneration. Human NP cells were obtained from patients with intervertebral disc degeneration undergoing surgical procedures in addition to ADSCs from liposuction patients. ADSC-derived extracellular nanovesicles were isolated and characterized. The differentiation and biological activity of NP cells cultured with or without ADSC-derived extracellular nanovesicles were assessed and inflammatory factors and intervertebral disc degeneration-associated markers were also measured. The results indicated that extracellular nanovesicles derived from ADSCs increased the migration and proliferation of NP cells and inhibited inflammatory activity, suggesting their utility for the treatment of intervertebral disc degeneration.
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2022
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Extracellular vesicles cargo from head and neck cancer cell lines disrupt dendritic cells function and match plasma microRNAs
Extracellular vesicles (EVs) are mediators of the immune system response. Encapsulated in EVs, microRNAs can be transferred between cancer and immune cells. To define the potential effects of EVs originated from squamous cell carcinoma cells on immune system response, we performed microRNA profiling of EVs released from two distinct cell lines and treated dendritic cells derived from circulating monocytes (mono-DCs) with these EVs. We confirmed the internalization of EVs by mono-DCs and the down-regulation of microRNA mRNA targets in treated mono-DCs. Differences in surface markers of dendritic cells cultivated in the presence of EVs indicated that their content disrupts the maturation process. Additionally, microRNAs known to interfere with dendritic cell function, and detected in EVs, matched microRNAs from squamous cell carcinoma patients’ plasma: miR-17-5p in oropharyngeal squamous cell carcinoma, miR-21 in oral squamous cell carcinoma, miR-16, miR-24, and miR-181a circulating in both oral and oropharyngeal squamous cell carcinoma, and miR-23b, which has not been previously described in plasma of head and neck squamous cell carcinoma, was found in plasma from patients with these cancer subtypes. This study contributes with insights on EVs in signaling between cancer and immune cells in squamous cell carcinoma of the head and neck.
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2022
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A Preclinical Investigation of GBM-N019 as a Potential Inhibitor of Glioblastoma via Exosomal mTOR/CDK6/STAT3 Signaling
Glioblastoma (GBM) is one of the most aggressive brain malignancies with high incidences of developing treatment resistance, resulting in poor prognoses. Glioma stem cell (GSC)-derived exosomes are important players that contribute to GBM tumorigenesis and aggressive properties. Herein, we investigated the inhibitory roles of GBM-N019, a novel small molecule on the transfer of aggressive and invasive properties through the delivery of oncogene-loaded exosomes from GSCs to naïve and non-GSCs. Our results indicated that GBM-N019 significantly downregulated the expressions of the mammalian target of rapamycin (mTOR), signal transducer and activator of transcription 3 (STAT3), and cyclin-dependent kinase 6 (CDK6) signaling networks with concomitant inhibitory activities against viability, clonogenicity, and migratory abilities of U251 and U87MG cells. Treatments with GBM-N019 halted the exosomal transfer of protein kinase B (Akt), mTOR, p-mTOR, and Ras-related protein RAB27A to the naïve U251 and U87MG cells, and rescued the cells from invasive and stemness properties that were associated with activation of these oncogenes. GBM-N019 also synergized with and enhanced the anti-GBM activities of palbociclib in vitro and in vivo. In conclusion, our results suggested that GBM-N019 possesses good translational relevance as a potential anti-glioblastoma drug candidate worthy of consideration for clinical trials against recurrent glioblastomas.
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2022
nm
Shell properties and concentration stability of acoustofluidic delivery agents
This paper investigates the shell elastic properties and the number-concentration stability of a new acoustofluidic delivery agent liposome in comparison to Definity™, a monolayer ultrasonic contrast agent microbubble. The frequency dependent attenuation of an acoustic beam passing through a microbubble suspension was measured to estimate the shell parameters. The excitation voltage was adjusted to ensure constant acoustic pressure at all frequencies. The pressure was kept at the lowest possible magnitude to ensure that effects from nonlinear bubble behaviour which are not considered in the analytical model were minimal. The acoustofluidic delivery agent shell stiffness Sp and friction Sf parameters were determined as (Sp = 0.11 N/m, Sf = 0.31 × 10−6 Kg/s at 25 °C) in comparison to the Definity™ monolayer ultrasound contrast agent which were (Sp = 1.53 N/m, Sf = 1.51 × 10−6 Kg/s at 25 °C). When the temperature was raised to physiological levels, the friction coefficient Sf decreased by 28% for the monolayer microbubbles and by only 9% for the liposomes. The stiffness parameter Sp of the monolayer microbubble decreased by 23% while the stiffness parameter of the liposome increased by a similar margin (27%) when the temperature was raised to 37 °C. The size distribution of the bubbles was measured using Tunable Resistive Pulse Sensing (TRPS) for freshly prepared microbubbles and for bubble solutions at 6 h and 24 h after activation to investigate their number-concentration stability profile. The liposome maintained >80% of their number-concentration for 24 h at physiological temperature, while the monolayer microbubbles maintained only 27% of their number-concentration over the same period. These results are important input parameters for the design of effective acoustofluidic delivery systems using the new liposomes.
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2022
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Circulating extracellular vesicles of patients with steroid-sensitive nephrotic syndrome have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes
Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (NS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with steroid-sensitive NS in relapse and remission, 10 healthy controls, and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry, and Western blot analysis. Major proteins from plasma EVs were identified via mass spectrometry. Gene Ontology classification analysis and Ingenuity Pathway Analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively expressed proteins that involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared with remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho-p38 and decreased the levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the NS phenotype in podocytes in vitro.NEW & NOTEWORTHY Up to now, the role of extracellular vesicles (EVs) in the pathogenesis of steroid-sensitive nephrotic syndrome (NS) has not been studied. Here, we found that relapse NS EVs contain significantly increased active RAC1, induce enhanced podocyte motility, and increase expression of RAC-GTP and phospho-p38 expression in vitro. These results suggest that plasma EVs are biologically active molecules in the pathogenesis of NS.
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2022
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Mesenchymal Stem Cells Do Not Lose Direct Labels Including Iron Oxide Nanoparticles and DFO-89Zr Chelates through Secretion of Extracellular Vesicles
Rapidly ageing populations are beset by tissue wear and damage. Stem cell-based regenerative medicine is considered a solution. Years of research point to two important aspects: (1) the use of cellular imaging to achieve sufficient precision of therapeutic intervention, and the fact that (2) many therapeutic actions are executed through extracellular vesicles (EV), released by stem cells. Therefore, there is an urgent need to interrogate cellular labels in the context of EV release. We studied clinically applicable cellular labels: superparamagnetic iron oxide nanoparticles (SPION), and radionuclide detectable by two main imaging modalities: MRI and PET. We have demonstrated effective stem cell labeling using both labels. Then, we obtained EVs from cell cultures and tested for the presence of cellular labels. We did not find either magnetic or radioactive labels in EVs. Therefore, we report that stem cells do not lose labels in released EVs, which indicates the reliability of stem cell magnetic and radioactive labeling, and that there is no interference of labels with EV content. In conclusion, we observed that direct cellular labeling seems to be an attractive approach to monitoring stem cell delivery, and that, importantly, labels neither locate in EVs nor affect their basic properties.
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2022
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Panax ginseng-Derived Extracellular Vesicles Facilitate Anti-Senescence Effects in Human Skin Cells: An Eco-Friendly and Sustainable Way to Use Ginseng Substances
Ginseng is a traditional herbal medicine in eastern Asian countries. Most active constituents in ginseng are prepared via fermentation or organic acid pretreatment. Extracellular vesicles (EVs) are released by most organisms from prokaryotes to eukaryotes and play central roles in intra- and inter-species communications. Plants produce EVs upon exposure to microbes; however, their direct functions and utility for human health are barely known, except for being proposed as delivery vehicles. In this study, we isolated EVs from ginseng roots (GrEVs) or the culture supernatants of ginseng cells (GcEVs) derived from Panax ginseng C.A. Meyer and investigated their biological effects on human skin cells. GrEV or GcEV treatments improved the replicative senescent or senescence-associated pigmented phenotypes of human dermal fibroblasts or ultraviolet B radiation-treated human melanocytes, respectively, by downregulating senescence-associated molecules and/or melanogenesis-related proteins. Based on comprehensive lipidomic analysis using liquid chromatography mass spectrometry, the lipidomic profile of GrEVs differed from that of the parental root extracts, showing significant increases in 70 of 188 identified lipid species and prominent increases in diacylglycerols, some phospholipids (phosphatidylcholine, phosphatidylethanolamine, lysophosphatidylcholine), and sphingomyelin, revealing their unique vesicular properties. Therefore, our results imply that GEVs represent a novel type of bioactive and sustainable nanomaterials that can be applied to human tissues for improving tissue conditions and targeted delivery of active constituents.
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2022
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miR‐146b Protects the Perinatal Brain against Microglia‐Induced Hypomyelination
OBJECTIVES: In the premature newborn, perinatal inflammation mediated by microglia contributes significantly to neurodevelopmental injuries including white matter injury (WMI). Brain inflammation alters development through neuroinflammatory processes mediated by activation of homeostatic microglia toward a pro-inflammatory and neurotoxic phenotype. Investigating immune regulators of microglial activation is crucial to find effective strategies to prevent and treat WMI. METHODS: Ex vivo microglial cultures and a mouse model of WMI induced by perinatal inflammation (interleukin-1-beta [IL-1β] and postnatal days 1-5) were used to uncover and elucidate the role of microRNA-146b-5p in microglial activation and WMI. RESULTS: A specific reduction in vivo in microglia of Dicer, a protein required for microRNAs maturation, reduces pro-inflammatory activation of microglia and prevents hypomyelination in our model of WMI. Microglial miRNome analysis in the WMI model identified miRNA-146b-5p as a candidate modulator of microglial activation. Ex vivo microglial cell culture treated with the pro-inflammatory stimulus lipopolysaccharide (LPS) led to overexpression of immunomodulatory miRNA-146b-5p but its drastic reduction in the microglial extracellular vesicles (EVs). To increase miRNA-146b-5p expression, we used a 3DNA nanocarrier to deliver synthetic miRNA-146b-5p specifically to microglia. Enhancing microglial miRNA-146b-5p overexpression significantly decreased LPS-induced activation, downregulated IRAK1, and restored miRNA-146b-5p levels in EVs. In our WMI model, 3DNA miRNA-146b-5p treatment significantly prevented microglial activation, hypomyelination, and cognitive defect induced by perinatal inflammation. INTERPRETATIONS: These findings support that miRNA-146b-5p is a major regulator of microglia phenotype and could be targeted to reduce the incidence and the severity of perinatal brain injuries and their long-term consequences. ANN NEUROL 2022;91:48-65.
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2022
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Exosomes from primed MSCs can educate monocytes as a cellular therapy for hematopoietic acute radiation syndrome
Background Acute radiation syndrome (ARS) is caused by acute exposure to ionizing radiation that damages multiple organ systems but especially the bone marrow (BM). We have previously shown that human macrophages educated with exosomes from human BM-derived mesenchymal stromal cells (MSCs) primed with lipopolysaccharide (LPS) prolonged survival in a xenogeneic lethal ARS model. The purpose of this study was to determine if exosomes from LPS-primed MSCs could directly educate human monocytes (LPS-EEMos) for the treatment of ARS. Methods Human monocytes were educated by exosomes from LPS-primed MSCs and compared to monocytes educated by unprimed MSCs (EEMos) and uneducated monocytes to assess survival and clinical improvement in a xenogeneic mouse model of ARS. Changes in surface molecule expression of exosomes and monocytes after education were determined by flow cytometry, while gene expression was determined by qPCR. Irradiated human CD34+ hematopoietic stem cells (HSCs) were co-cultured with LPS-EEMos, EEMos, or uneducated monocytes to assess effects on HSC survival and proliferation. Results LPS priming of MSCs led to the production of exosomes with increased expression of CD9, CD29, CD44, CD146, and MCSP. LPS-EEMos showed increases in gene expression of IL-6, IL-10, IL-15, IDO, and FGF-2 as compared to EEMos generated from unprimed MSCs. Generation of LPS-EEMos induced a lower percentage of CD14+ monocyte subsets that were CD16+, CD73+, CD86+, or CD206+ but a higher percentage of PD-L1+ cells. LPS-EEMos infused 4 h after lethal irradiation significantly prolonged survival, reducing clinical scores and weight loss as compared to controls. Complete blood counts from LPS-EEMo-treated mice showed enhanced hematopoietic recovery post-nadir. IL-6 receptor blockade completely abrogated the radioprotective survival benefit of LPS-EEMos in vivo in female NSG mice, but only loss of hematopoietic recovery was noted in male NSG mice. PD-1 blockade had no effect on survival. Furthermore, LPS-EEMos also showed benefits in vivo when administered 24 h, but not 48 h, after lethal irradiation. Co-culture of unprimed EEMos or LPS-EEMos with irradiated human CD34+ HSCs led to increased CD34+ proliferation and survival, suggesting hematopoietic recovery may be seen clinically. Conclusion LPS-EEMos are a potential counter-measure for hematopoietic ARS, with a reduced biomanufacturing time that facilitates hematopoiesis.
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2022
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Extracellular Vesicles from Thapsigargin-Treated Mesenchymal Stem Cells Ameliorated Experimental Colitis via Enhanced Immunomodulatory Properties
Therapeutic applications of extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) have attracted considerable attention because of their immunomodulatory properties against immune-mediated, inflammatory diseases. Here, we demonstrated enhanced immunomodulatory properties of EVs secreted from endoplasmic reticulum (ER) stress inducer thapsigargin (TSG)-primed human Wharton's jelly-derived MSCs (WJ-MSCs). EVs from TSG-primed WJ-MSCs (TSG-EV) showed increased yield and expression of immunomodulatory factors, such as transforming growth factor-β1 (TGFβ), cyclooxygenase-2 (COX2), and especially indoleamine 2,3-dioxygenase (IDO), compared to control EVs. TSG-EV showed a significantly enhanced immunosuppressive effect on human peripheral blood-derived T cell proliferation and Th1 and Th17 differentiation, whereas Treg and M2-type macrophage were enriched compared to a control EV-treated group. Furthermore, TSG-EV substantially mitigated mouse experimental colitis by reducing the inflammatory response and maintaining intestinal barrier integrity. A significant increase of Tregs and M2-type macrophages in colitic colons of a TSG-EV-treated mouse suggests an anti-inflammatory effect of TSG-EV in colitis model, possibly mediated by Treg and macrophage polarization. These data indicate that TSG treatment promoted immunomodulatory properties of EVs from WJ-MSCs, and TSG-EV may provide a new therapeutic approach for treatment of colitis.
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2022
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Involvement of the Choroid Plexus in the Pathogenesis of Niemann-Pick Disease Type C
Niemann-Pick type C (NPC) disease, sometimes called childhood Alzheimer's, is a rare neurovisceral lipid storage disease with progressive neurodegeneration leading to premature death. The disease is caused by loss-of-function mutations in the Npc1 or Npc2 gene which both result into lipid accumulation in the late endosomes and lysosomes. Since the disease presents with a broad heterogenous clinical spectrum, the involved disease mechanisms are still incompletely understood and this hampers finding an effective treatment. As NPC patients, who carry NPC1 mutations, have shown to share several pathological features with Alzheimer's disease (AD) and we and others have previously shown that AD is associated with a dysfunctionality of the blood-cerebrospinal fluid (CSF) barrier located at choroid plexus, we investigated the functionality of this latter barrier in NPC1 pathology. Using NPC1-/- mice, we show that despite an increase in inflammatory gene expression in choroid plexus epithelial (CPE) cells, the blood-CSF barrier integrity is not dramatically affected. Interestingly, we did observe a massive increase in autophagosomes in CPE cells and enlarged extracellular vesicles (EVs) in CSF upon NPC1 pathology. Additionally, we revealed that these EVs exert toxic effects on brain tissue, in vitro as well as in vivo. Moreover, we observed that EVs derived from the supernatant of NPC1-/- choroid plexus explants are able to induce typical brain pathology characteristics of NPC1-/-, more specifically microgliosis and astrogliosis. Taken together, our data reveal for the first time that the choroid plexus and CSF EVs might play a role in the brain-related pathogenesis of NPC1.
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2022
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Uptake of circulating extracellular vesicles from rectal cancer patients and differential responses by human monocyte cultures
Extracellular vesicles (EVs) released by tumor cells can directly or indirectly modulate the phenotype and function of the immune cells of the microenvironment locally or at distant sites. The uptake of circulating EVs and the responses by human monocytes in vitro may provide new insights into the underlying biology of the invasive and metastatic processes in cancer. Although a mixed population of vesicles is obtained with most isolation techniques, we predominantly isolated exosomes (small EVs) and microvesicles (medium EVs) from the SW480 colorectal cancer cell line (established from a primary adenocarcinoma of the colon) by sequential centrifugation and ultrafiltration, and plasma EVs were prepared from 22 patients with rectal adenoma polyps or invasive adenocarcinoma by size-exclusion chromatography. The EVs were thoroughly characterized. The uptake of SW480 EVs was analyzed, and small SW480 EVs were observed to be more potent than medium SW480 EVs in inducing monocyte secretion of cytokines. The plasma EVs were also internalized by monocytes; however, their cytokine-releasing potency was lower than that of the cell line-derived vesicles. The transcriptional changes in the monocytes highlighted differences between adenoma and adenocarcinoma patient EVs in their ability to regulate biological functions, whereas the most intriguing changes were found in monocytes receiving EVs from patients with metastatic compared with localized cancer.
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2022
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Percutaneous Intracoronary Delivery of Plasma Extracellular Vesicles Protects the Myocardium Against Ischemia-Reperfusion Injury in Canis
Plasma circulating extracellular vesicles (EVs) have been utilized as a potential therapeutic strategy to treat ischemic disease through intramyocardial injection (efficient but invasive) or tail vein injection (noninvasive but low cardiac retention). An effective and noninvasive delivery of EVs for future clinical use is necessary. The large animal (canine) model was complemented with a murine ischemia-reperfusion injury (IRI) model, as well as H9 human embryonic stem cell–induced cardiomyocytes or neonatal rat cardiomyocytes to investigate the effective delivery method and the role of plasma EVs in the IRI model. We further determine the crucial molecule within EVs that confers the cardioprotective role in vivo and in vitro and investigate the efficiency of CHP (cardiac homing peptide)-linked EVs in alleviating IRI. D-SPECT imaging showed that percutaneous intracoronary delivery of EVs reduced infarct extent in dogs. CHP-EVs further reduced IRI-induced cardiomyocyte apoptosis in mice and neonatal rat cardiomyocytes. Mechanistically, administration of EVs by percutaneous intracoronary delivery (in dog) and myocardial injection (in mice) just before reperfusion reduced infarct size of IRI by increasing miR-486 levels. miR-486–deleted EVs exacerbated oxygen-glucose deprivation/reoxygenation–induced human embryonic stem cell–induced cardiomyocytes and neonatal rat cardiomyocyte apoptosis. EV-miR-486 inhibited the PTEN (phosphatase and tensin homolog deleted on chromosome ten) expression and then promoted AKT (protein kinase B) activation in human embryonic stem cell–induced cardiomyocytes and neonatal rat cardiomyocytes. In conclusion, plasma-derived EVs convey miR-486 to the myocardium and attenuated IRI-induced infarction and cardiomyocyte apoptosis. CHP strategy was effective to improve cardiac retention of EVs in mice (in vivo) and dogs (ex vivo).
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2022
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Parasite worm antigens instruct macrophages to release immunoregulatory extracellular vesicles
Emerging evidence suggests that immune cells not only communicate with each other through cytokines, chemokines, and cell surface receptors, but also by releasing small membranous structures known as extracellular vesicles (EVs). EVs carry a variety of different molecules that can be taken up by recipient cells. Parasitic worms are well known for their immunomodulatory properties, but whether they can affect immune responses by altering EV-driven communication between host immune cells remains unclear. Here we provide evidence that stimulation of bone marrow-derived macrophages (BMDMs) with soluble products of Trichuris suis (TSPs), leads to the release of EVs with anti-inflammatory properties. Specifically, we found that EVs from TSP-pulsed BMDMs, but not those from unstimulated BMDMs can suppress TNFα and IL-6 release in LPS-stimulated BMDMs and BMDCs. However, no polarization toward M1 or M2 was observed in macrophages exposed to EVs. Moreover, EVs enhanced reactive oxygen species (ROS) production in the exposed BMDMs, which was associated with a deregulated redox homeostasis as revealed by pathway analysis of transcriptomic data. Proteomic analysis identified cytochrome p450 (CYP450) as a potential source of ROS in EVs from TSP-pulsed BMDMs. Finally, pharmacological inhibition of CYP450 activity could suppress ROS production in those BMDMs. In summary, we find that TSPs can modulate immune responses not only via direct interactions but also indirectly by eliciting the release of EVs from BMDMs that exert anti-inflammatory effects on recipient cells.
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2022
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Single‐step equipment‐free extracellular vesicle concentration using super absorbent polymer beads
Extracellular vesicles (EVs) contain useful biomarkers for disease diagnosis and are promising biomaterials for the delivery of therapeutic molecules in vivo. Accordingly, an efficient concentration method is necessary for large-scale production or high-throughput isolation of EVs from bulk liquid samples, including culture medium and body fluids, to achieve their clinical application. However, current EV concentration methods, including ultrafiltration, are limited with respect to cost, efficiency, and centrifugation time. In this study, we developed the first single-step, equipment-free EV concentration method using super absorbent polymer (SAP) beads. SAP beads absorb small molecules, including water, via nano-sized channels but expel and thereby concentrate EVs. Consequently, the beads drastically enrich EVs by reducing the solution volume in a single step, without affecting EV characteristics. Moreover, the purity of the concentrated EV solution was high due to the absorption of protein impurities by SAP beads. To further demonstrate the versatility of the method, we showed that SAP beads successfully enrich EVs in human urine samples and culture medium, enabling better isolation performance than conventional ultrafiltration. We believe the newly developed approach and insight gained in this study will facilitate the use of EVs as prominent biomaterials for disease diagnosis and therapy.
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2022
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Oviduct as a sensor of embryo quality: deciphering the extracellular vesicle (EV)-mediated embryo-maternal dialogue
Embryo-derived extracellular vesicles (EVs) may play a role in mediating the embryo-maternal dialogue at the oviduct, potentially carrying signals reflecting embryo quality. We investigated the effects of bovine embryo-derived EVs on the gene expression of bovine oviductal epithelial cells (BOECs), and whether these effects are dependent on embryo quality. Presumptive zygotes were cultured individually in vitro in culture medium droplets until day 8 while their development was assessed at day 2, 5 and 8. Conditioned medium samples were collected at day 5 and pooled based on embryo development (good quality embryo media and degenerating embryo media). EVs were isolated from conditioned media by size exclusion chromatography and supplemented to primary BOEC monolayer cultures to evaluate the effects of embryo-derived EVs on gene expression profile of BOEC. Gene expression was quantified by RNA-seq and RT-qPCR. A total of 7 upregulated and 18 downregulated genes were detected in the BOECs supplemented with good quality embryo-derived EV compared to the control. The upregulated genes included interferon-τ-induced genes, such as OAS1Y, MX1 and ISG15, which have previously been reported as upregulated in the oviductal epithelial cells in the presence of embryos. Of the upregulated genes, OAS1Y and MX1 were validated with RT-qPCR. In contrast, only one differentially expressed gene was detected in BOECs in response to degenerating embryo-derived EVs, suggesting that oviductal responses are dependent on embryo quality. Our results support the hypothesis that embryo-derived EVs are involved in embryo-maternal communication at the oviduct and the oviductal response is dependant on the embryo quality.Key messages• Extracellular vesicles (EVs) released by individually cultured pre-implantation bovine embryos can alter the gene expression of primary oviductal epithelial cells.• The oviductal response, in terms of gene expression, to the bovine embryo-derived EVs varied depending on the embryo quality.• In vivo, the oviduct may have the ability to sense the quality of the pre-implantation embryos.• The observed effect of embryo-derived EVs on oviductal epithelial cells could serve as a non-invasive method of evaluating the embryo quality.
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2022
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Dynamics of SARS-CoV-2 Spike Proteins in Cell Entry: Control Elements in the Amino-Terminal Domains
Selective pressures drive adaptive changes in the coronavirus spike proteins directing virus-cell entry. These changes are concentrated in the amino-terminal domains (NTDs) and the receptor-binding domains (RBDs) of complex modular spike protein trimers. The impact of this hypervariability on virus entry is often unclear, particularly with respect to sarbecovirus NTD variations. Therefore, we constructed indels and substitutions within hypervariable NTD regions and used severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus-like particles and quantitative virus-cell entry assays to elucidate spike structures controlling this initial infection stage. We identified NTD variations that increased SARS-CoV-2 spike protein-mediated membrane fusion and cell entry. Increased cell entry correlated with greater presentation of RBDs to ACE2 receptors. This revealed a significant allosteric effect, in that changes within the NTDs can orient RBDs for effective virus-cell binding. Yet, those NTD changes elevating receptor binding and membrane fusion also reduced interdomain associations, leaving spikes on virus-like particles susceptible to irreversible inactivation. These findings parallel those obtained decades ago, in which comparisons of murine coronavirus spike protein variants established inverse relationships between membrane fusion potential and virus stability. Considerable hypervariability in the SARS-CoV-2 spike protein NTDs also appear to be driven by counterbalancing pressures for effective virus-cell entry and durable extracellular virus infectivity. These forces may selectively amplify SARS-CoV-2 variants of concern. IMPORTANCE Adaptive changes that increase SARS-CoV-2 transmissibility may expand and prolong the coronavirus disease 2019 (COVID-19) pandemic. Transmission requires metastable and dynamic spike proteins that bind viruses to cells and catalyze virus-cell membrane fusion. Using newly developed assays reflecting these two essential steps in virus-cell entry, we focused on adaptive changes in SARS-CoV-2 spike proteins and found that deletions in amino-terminal domains reset spike protein metastability, rendering viruses less stable yet more poised to respond to cellular factors that prompt entry and subsequent infection. The results identify adjustable control features that balance extracellular virus stability with facile virus dynamics during cell entry. These equilibrating elements warrant attention when monitoring the evolution of pandemic coronaviruses.
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2022
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