Nanoparticle Tracking Analysis Research Articles

The proteomic landscape of extracellular vesicles derived from human intervertebral disc cells.

Extracellular vesicles (EVs) function as biomarkers and are crucial in cell communication and regulation, with therapeutic potential for intervertebral disc (IVD)-related low back pain (LBP). EV cargo is often affected by tissue health, which may affect the therapeutic potential. There is currently limited knowledge of how the cargo of IVD cell-derived EVs varies with tissue health and how differences in proteomic profile affect the predicted biological functions. Our study purified EVs from human IVD cell conditioned media by size-exclusion chromatography. Nanoparticle tracking analysis was conducted to measure EV size and concentration. Transmission electron microscopy and Western blot were performed to examine EV structure and markers. Tandem mass tag-mass spectrometry was conducted to determine protein cargo. Most EVs were exosomes and intermediate microvesicles with an increasing amount linked to disease progression. Of the proteins detected, 88.6% were shared across the non-degenerate, mildly-degenerate, and degenerate samples. GO and KEGG analyses revealed that cargo from the mildly-degenerate samples was the most distinct, with the proteins in high abundance strongly associated with extracellular matrix (ECM) organization and structure. Shared proteins, highly expressed in the non-degenerate and degenerate samples, showed strong associations with cell adhesion, ECM-receptor interaction, and vesicle-mediated transport, respectively. Our findings indicate that EVs from IVD cells from tissue with different degrees of degeneration share a majority of the cargo proteins. However, the level of expression differs with degeneration grade. Cargo from the mildly-degenerate samples exhibits the most differences. A better understanding of changes in EV cargo in the degenerative process may provide novel information related to molecular mechanisms underlying IVD degeneration and suggest new potential treatment modalities for IVD-related LBP.

Extracellular Vesicles from Lung Adenocarcinoma Cells Induce Activation of Different Cancer-Associated Fibroblast Subtypes

Background: Lung cancer, including the major subtype lung adenocarcinoma (LUAD), is the leading cause of cancer deaths worldwide, largely due to metastasis. Improving survival rates requires new treatment strategies and a deeper understanding of the mechanisms that drive tumor progression within the tumor microenvironment (TME). This study investigated the impact of extracellular vesicles (EVs) derived from LUAD cells on lung fibroblasts. Methods: EVs were isolated from LUAD cell lines via ultracentrifugation and characterized using nanoparticle tracking analysis and Western blotting. Lung fibroblasts were treated with PBS, TGFβ, or EVs, and their activation was assessed through protein (Western blotting) and RNA analyses (RNA seq and RT-qPCR). Results: The results confirmed the TGFβ induced activation and showed that LUAD EVs could also activate fibroblasts, increasing cancer-associated fibroblast (CAF) markers. While EV-induced CAF activation displayed unique features, like an increase in proliferation-related genes, the EV and TGFβ treatments also shared some differentially expressed genes. The EV groups induced a higher expression of ECM remodeling and EMT-associated genes, but some of those genes were also upregulated in the TGFβ group. Mesenchymal genes POSTN and SPOCK1 were significantly upregulated in TGFβ- and EV-treated fibroblasts. Their secretion as proteins from the TGFβ- and EV-induced CAFs was not significant, confirmed through ELISA. Conclusions: These findings suggest that LUAD EVs play a role in CAF activation through both shared and distinct pathways compared to canonical TGFβ activation, potentially identifying novel gene expressions involved in CAF activation. Additionally, optimal protein secretion conditions of confirmed CAF-upregulated genes need to be established to determine their contribution to the TME.

Isolation of Small Extracellular Vesicles (sEVs) from the Apoplastic Wash Fluid of Nicotiana benthamiana Leaves.

Extracellular vesicles (EVs) are small membranous vesicles secreted by cells into their surrounding extracellular environment. Similar to mammalian EVs, plant EVs have emerged as essential mediators of intercellular communication in plants that facilitate the transfer of biological material between cells. They also play essential roles in diverse physiological processes including stress responses, developmental regulation, and defense mechanisms against pathogens. In addition, plant EVs have demonstrated promising health benefits as well as potential therapeutic effects in mammalian health. Despite the plethora of potential applications using plant EVs, their isolation and characterization remains challenging. In contrast to mammalian EVs, which benefit from more standardized isolation protocols, methods for isolating plant EVs can vary depending on the starting material used, resulting in diverse levels of purity and composition. Additionally, the field suffers from the lack of plant EV markers. Nevertheless, three main EV subclasses have been described from leaf apoplasts: tetraspanin 8 positive (TET8), penetration-1-positive (PEN1), and EXPO vesicles derived from exocyst-positive organelles (EXPO). Here, we present an optimized protocol for the isolation and enrichment of small EVs (sEVs; <200 nm) from the apoplastic fluid from Nicotiana benthamiana leaves by ultracentrifugation. We analyze the preparation through transmitted electron microscopy (TEM), nanoparticle tracking analysis (NTA), and western blotting. We believe this method will establish a basic protocol for the isolation of EVs from N. benthamiana leaves, and we discuss technical considerations to be evaluated by each researcher working towards improving their plant sEV preparations. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol: Isolation and enrichment of small extracellular vesicles (sEVs) from the apoplastic fluid of Nicotiana benthamiana leaves.

Effect of extracellular vesicles derived from inflammatory H9C2 cardiomyocytes on the polarization of macrophages and its mechanism

Abstract Objective The aim of this study was to investigate the effect of Extracellular vesicles (EVs) derived from inflammatory cardiomyocytes on the polarization of macrophages and its related mechanism. Methods EVs derived from the inflammatory and normal cardiomyocyte cell line H9C2 were extracted by differential centrifugation respectively. The extracted EVs were characterized by nanoparticle tracking analysis (NTA), transmission electron microscopy (TEM) and Western blot analysis (WB). The RAW264.7 macrophages in the inflammatory environment were treated with EVs, the proliferative activities were detected by CCK8, the expression of inflammatory factors were detected by qRT-PCR, and the differentiation of RAW264.7 macrophages into M1 and M2 was analyzed by flow cytometry. In vivo experiments, the animal model of viral myocarditis was established in Balb/c mice with intraperitoneal injection of CVB3. EVs derived from inflammatory H9C2 cardiomyocytes and medium were injected into mice by tail vein injection, respectively. The mice myocardium was stained by immunohistochemistry, and the total RNA of myocardial tissue was extracted for the detection of inflammatory factors by RT-PCR. In terms of mechanism research, mRNA expression profiles of RAW264.7 cells after EVs treatment were analyzed by RNA sequencing for functional annotation and mechanism study. At the same time, the extracted EVs derived from H9C2 normal group and the inflammatory group were subjected to 4D-Lable Free technology for quantitative proteomic analysis, and the results were analyzed bioinformatics, and then by WB analysis.Results We found that EVs derived from inflammatory H9C2 cardiomyocytes decreased the proliferative activity of RAW264.7 macrophages, the expression of pro-inflammatory cytokines IL-6, IL-1β, TNF-ɑ, and the proportion of M1 macrophages in the inflammatory state, while the proportion of M2 macrophages, which mainly inhibit inflammation, increased, and the expression levels of anti-inflammatory cytokines IL-10 and CD-206 increased. Results of immunohistochemical staining and RT-PCR showed that the EVs of inflammatory H9C2 regulated the heterogeneity of infiltrating macrophages and the expression of several inflammatory cytokines in mouse myocardium inflammatory tissue. Meanwhile, in vitro studies have found that the regulatory effect of EVs derived from inflammatory H9C2 cardiomyocytes on macrophage heterogeneity is mediated by MAPK signaling pathway. Conclusion EVs derived from inflammatory H9C2 cardiomyocytes can regulate the polarization of macrophages, change the expression of inflammatory factors, and regulate the myocardial inflammatory microenvironment, which may be achieved by mediating the p38 MAPK pathway through the delivery of PP2A.

Sex-specific differences in the severity of pulmonary hypoplasia in experimental congenital diaphragmatic hernia and implications for extracellular vesicle-based therapy.

Amniotic fluid stem cell extracellular vesicles (AFSC-EVs) hold regenerative potential to treat hypoplastic lungs secondary to congenital diaphragmatic hernia (CDH). This study aims to investigate sex-specific differences in pulmonary hypoplasia severity and responses to AFSC-EV administration in an experimental CDH mouse model. C57BL/6J dams were fed with nitrofen + bisdiamine (left-sided CDH) or olive oil only (control) at embryonic day (E) 8.5. Lungs were dissected (E18.5), grown ex vivo and treated with medium ± AFSC-EVs that were collected via ultracentrifugation and characterized (nanoparticle tracking analysis, electron microscopy, Western blotting). Pulmonary hypoplasia was assessed via mean linear intercept (MLI). Gene and protein expression changes (Cd31, Enos, Il1b, TNFa) were measured via RT-qPCR and immunofluorescence. Pups were genotyped for Sry. Experimental CDH showed a male predominance without sex differences for pulmonary hypoplasia severity, fetal lung vascularization, and inflammation. AFSC-EV administration led to improved lung growth (decreased MLI), improved fetal lung vascularization (increased Cd31 and Enos), and decreased fetal lung inflammation (Il1b, TNFa). There was no sex-specific response to AFSC-EV administration. This study shows sex-independent impaired lung growth, vascularization and fetal lung inflammation in a CDH mouse model. Antenatal administration of AFSC-EVs reverses aspects of pulmonary hypoplasia secondary to CDH independent of the biological sex.

Serum exosomal long non-coding RNA H19 as a theragnostic target for atrial fibrillation

Abstract Background/Introduction Exosomes are membrane vesicles of 30- to 150-nm secreted by most cell types that can mediate intercellular communication by transmitting various forms of RNAs. Long non-coding RNAs (lncRNAs, ≥ 200 nucleotides length) have been reported to play important roles in the pathophysiological processes that promote the development and progression of many diseases. Interestingly, recent studies have shown that exosomal lncRNAs exhibit different expression profiles in various diseases and are being extensively studied in the medical field as an ideal target for the diagnosis and treatment. However, there is still little known about their role in atrial fibrillation (AF). Purpose The aim of this study was to identify a novel theragnostic target exosomal lncRNA for AF. Methods First, serum exosomes from controls and patients with persistent AF were isolated and characterized by transmission electron microscopy, nanoparticle tracking analysis, and western blot. Next, serum exosomal lncRNA expression profiles were explored using RNA-sequencing analysis. In addition, the expression levels of candidate exosomal lncRNAs were confirmed using qRT-PCR (n = 50 per group). Finally, AF-related pathophysiological mechanisms of exosomal lncRNA were investigated in angiotensin II (Ang II)-treated human induced pluripotent stem cell-derived atrial cardiomyocytes (iPSC-atrial CMs). Results After RNA-sequencing analysis, we identified 27 differentially expressed lncRNAs (i.e., 4 upregulated and 23 downregulated lncRNAs with a |fold change| ≥ 2 and p &amp;lt; 0.05) in serum exosomes from patients with persistent AF compared with the controls. In fact, qRT-PCR reveled that exosomal lncRNA H19 was consistently downregulated in the serum of patients with persistent AF compared with the controls (p &amp;lt; 0.01). In addition, exosomal lncRNA H19 exhibited significant diagnostic validity for AF. Notably, we confirmed that exosomal lncRNA H19 was involved in AF-related pathophysiological mechanisms. In Ang II-treated iPSC-atrial CMs, inhibition of lncRNA H19 significantly aggravated Ang II-induced increases in levels of hypertrophic markers (ANP, BNP and β-MHC), cell surface area and inflammation (p &amp;lt; 0.05). Mechanistically, we found that loss of lncRNA H19 weakens the inhibition of its direct target microRNAs, miR-141-3p and miR-200a-3p, leading to downregulation of their target PTEN, thereby promoting the atrial remodeling and the consequent AF. Conclusion In conclusion, serum-derived exosomal lncRNA H19 could serve as a potential target for the diagnosis and treatment of AF.

Human coronary blood single-cell sequencing combined with exosome proteomic profiling reveals the molecular mechanism of recurrent in-stent restenosis (R-ISR)

Abstract Background Percutaneous coronary intervention (PCI) is recognized as the core treatment for symptomatic myocardial ischemia and acute coronary syndrome (ACS). However, the complex pathogenesis of recurrent in-stent restenosis (R-ISR) after stent implantation is still insufficiently understood. Purpose This study reports the first single-cell RNA sequencing and proteomic profiling to characterize pathogenesis of R-ISR in humans. Methods To reveal the pathophysiology and molecular mechanism of R-ISR, we performed single-cell RNA sequencing to identify RISR-induced changes in transcriptional landscape in coronary blood mononuclear cell composition. Besides, proteomic profiling of human coronary plasma-derived exosomes was used to further screen different proteins. Transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blot analysis were performed to identify exosomes. In vitro cell biology experiments were used to validate the discoveries of bioinformatics analysis. Results Compared with normal subjects, the percentage of FCGR3B high monocytes, memory T cells and plasmacytoid dendritic cells (DCs) was increased in R-ISR. Furthermore, we observed a marked increase in the expression of JUN, FOS, CXCL8, S100A8 enriched in the IL-17 signaling pathway that is known to play a crucial role in activating inflammation, mediating immune response, and even maintaining inflammation to chronicity. According to in vitro experiments, the co-cultured system demonstrated that FCGR3B high monocytes isolated from RISR patients activated the expression of inflammatory factors in endothelial cells (ECs), such as IL-1β, IL-6 and TNF-α. Inflammatory activated endothelial cells could release a large amount of exosomes, proteomic profiling of exosomes isolated from ECs observed a significant upregulation of MYLK expression, which could promote proliferation and migration of vascular smooth muscle cells (VSMCs) by activating VEGFR-2 signaling pathway. Conclusions Our study reveals a novel subset of FCGR3B high monocytes in R-ISR patients that could activate ECs inflammation via IL-17 signaling pathway. Activated ECs release exosomes rich in MYLK, which promote proliferation and migration of VSMCs, ultimately leading to repeated restenosis.

Extracellular vesicles characterization in patients with hypertrophic cardiomyopathy

Abstract Background Hypertrophic cardiomyopathy (HCM) is a genetic disorder, diagnosed according to the presence of phenotypical traits of the myocardium. A specific biomarker which can associate with the severity of HCM is lacking. Extracellular Vesicles (EVs), nanoparticles released by cells into biological fluids, hold promise as accessible diagnostic tools, as their abundance and molecular composition can reflect the severity of cardiac diseases (Rizzuto 2024). We aim at characterising plasma-derived EVs isolated from 28 HCM patients and 18 healthy volunteers (CTR). Methods The whole cohort underwent transthoracic echocardiography, whereas magnetic resonance and exome sequencing was performed on HCM patients. EVs were isolated via ultracentrifugation from plasma of both groups. Quantitative and qualitative assessments of EVs were performed by nanoparticle tracking analysis, transmission electron microscopy, Western blot, targeted (Olink) and untargeted (nLC-MS/MS) proteomics and FACS analysis. Plasma-derived EV subpopulations were characterised according to their cellular origin. Data are expressed as median and interquartile range (25th, 75th). Results Most patients were male (68% HCM and 66% CTR) with a median age of 58 (49-69) years (HCM) and 47 (44-52) (CTR). Among HCM patients, missense mutations in the MYH7 gene were the most identified. The median maximum wall thickness (WTMax) in HCM was 16 mm (15-19) vs 8 mm (7-9) in CTR. The isolated EVs expressed tetraspanins (CD9, CD63 and CD81), Alix and β-integrin and showed an intact phospholipid bilayer of 100 nm in size. EV concentration was reduced in HCM vs CTRL, respectively 2.8*109 EV/ml/cell count (2*109-4*109) and 4.6*10⁹ EV/ml/cell count (3*109-6*109). Among 15 plasma-derived EV subpopulations studied, those released from platelets (CD41a) and activated platelets (CD62P and CD40L) were increased in HCM patients vs CTR by 1.7 fold. Negative associations were found between E/e’, parameter of diastolic function, and cardiomyocyte-derived EVs (r= -0.2658), and between left ventricle ejection fraction and platelet-derived EVs (r=-0.2189); a positive correlation was found between WTMax and EVs derived from activated endothelial cells (r=0.2330). The proteomic analysis of EVs shows an enrichment in proteins related to platelets adhesion and inflammation in HCM patients. Conclusions HCM patients present a peculiar phenotypic pattern of EVs that may associate with diagnostic clinical features of HCM.

Extracellular vesicles of Clonorchis sinensis promote the malignant phenotypes of cholangiocarcinoma via NF-κB/EMT axis.

Clonorchis sinensis infection is an important risk factor for cholangiocarcinoma (CCA). It has been reported that extracellular vesicles (EVs) are involved in the parasite-host interaction, and EVs of C. sinensis (CsEVs) can contribute to biliary injuries and inflammation. However, uncertainty surrounds the function of CsEVs in the progression of CCA. In this study, differential ultracentrifugation was used to separate CsEVs from the culture supernatant of C. sinensis adult worms, and they were then identified by transmission electron microscopy, nanoparticle tracking analysis and proteome assays. CCK8, EdU-488 and colony formation assays were used to explore the effect of CsEVs on the proliferation of CCA cells in vitro. Wound healing assays, transwell assays and in vivo lung metastasis model were conducted to evaluate the migration and invasion abilities. Moreover, the involvement of EMT process, as well as NF-κB and ERK signaling pathway was assessed. Results showed that CsEVs were successfully isolated and could be taken up by CCA cells, which promoted proliferation by accelerating cell cycle progression. In addition, CsEVs could facilitate cell metastasis by triggering the epithelial-mesenchymal transition (EMT). Mechanistically, activation of NF-κB signaling pathway was involved in the CsEVs-mediated EMT, which could be reversed partly by BAY 11-7082 (an inhibitor of NF-κB). In conclusion, these findings suggested that CsEVs could induce the aberrant proliferation and metastasis of CCA cells by stimulating the NF-κB/EMT axis, providing a novel theoretical explanation for liver fluke-associated CCA.

Salivary Extracellular Vesicles Separation: Analysis of Ultracentrifugation-Based Protocols.

The clinical potential of extracellular vesicles (EVs) is widely acknowledged, yet the standardization and reproducibility of its separation remain challenging. This study compares three protocols: ultracentrifugation (UC), UC with purification step (UC + PS), and a combined protocol using polymer-based precipitation and UC (PBP + UC). Salivary samples were collected from healthy donors. EVs were separated (UC, UC + PS, and PBP + UC) and characterized using transmission electron microscopy, nanoparticle tracking analysis, EV purity, RNA concentration, and Western blotting. miRNA expression was evaluated by quantitative RT-PCR. Statistical analyses comparing groups were performed using ANOVA. All methods successfully separated CD9+ and CD63+ EVs from saliva. The UC + PS and PBP + UC protocols yielded the highest concentrations of EVs, enriched in < 200 nm vesicles. EV purity and RNA recovery were comparable among all methods. Expression of miR-16, miR-27a, and miR-99a was successfully detected using all methods. The UC + PS and PBP + UC protocols demonstrate comparable efficiency in separating salivary EVs. However, the combined PBP + UC protocol, with its simplified processing capability, offers a significant advantage, particularly in the initial phase of EV separation. This finding suggests its potential application in clinical settings where time-sensitive simple processing is critical. Further validation is needed to confirm its effectiveness for transcriptomic and proteomic analyses.

Phosphorylated tau in cerebrospinal fluid-derived extracellular vesicles in Alzheimer’s disease: a pilot study

Alzheimer’s disease (AD) is a debilitating neurodegenerative disorder characterized by brain aggregation of β-amyloid (Aβ) peptides and phosphorylated tau (P-tau) proteins. Extracellular vesicles (EVs) can be isolated and studied for potential roles in disease. While several studies have tested plasma-derived EVs in AD, few have analyzed EVs from cerebrospinal fluid (CSF), which are potentially more closely related to brain changes. This study included 20 AD patients and 20 cognitively unimpaired (CU) participants. Using a novel EV isolation method based on acoustic trapping, we isolated and purified EVs from minimal CSF volumes. EVs were lysed and analyzed by immunoassays for P-tau217 and P-tau181. Isolation was confirmed through transmission electron microscopy and the presence of EV-specific markers (CD9, CD63, CD81, ATP1A3). Nanoparticle tracking analysis revealed a high variance in EV distribution. AD patients exhibited increased P-tau181 and decreased P-tau217 in EVs, leading to a higher EV P-tau181/P-tau217 ratio compared to CU. No significant differences in EV counts or sizes were observed between AD and CU groups. This study is the first to use acoustic trapping to isolate EVs from CSF and demonstrates differential P-tau content in AD-derived EVs, warranting further research to understand the relationship between these EV changes and brain pathology.

Characterization of Extracellular Vesicles by Sulfophosphovanillin Colorimetric Assay and Raman Spectroscopy.

Detailed characterization of extracellular vesicles (EVs) is crucial for their application in medical diagnostics. However, the complexity of their chemical composition and the heterogeneity of EV populations make their characterization challenging. Here we describe two analytical procedures that can help overcome this challenge. Small EVs were isolated from conditioned cell culture media using ultracentrifugation and characterized using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Raman spectroscopy was used to assess the overall composition of the isolated samples and lipids extracted from them. Sulfophosphovanillin (SPV) colorimetric assay was used to quantify the contents of lipid. Six samples of EVs were characterized. The lipid contents measured using SPV assay was in reasonable agreement with the quantitative estimates based on the particle size and concentration measured using NTA. The most peaks observed in the Raman spectra could be attributed to either proteins or lipids, and their origins was confirmed by lipid extraction. The protein-to-lipid ratio was estimated based on the Raman spectra. The experiential procedures described in this study will help to overcome the challenge of quick and highly informative characterization of the EVs.

Propionibacterium freudenreichii MJ2-derived extracellular vesicles inhibit RANKL-induced osteoclastogenesis and improve collagen-induced rheumatoid arthritis

Rheumatoid arthritis causes excessive bone loss by stimulating osteoclast differentiation. Extracellular vesicles are valuable disease markers, conveyors of distant cell-to-cell communication, and carriers for drug delivery. The aim of this study was to investigate the anti-osteoclastogenic effects of extracellular vesicles derived from dairy Propionibacterium freudenreichii MJ2 (PFEVs) and the improvement effect of PFEVs on collagen-induced arthritis (CIA) animal model. PFEVs were observed by scanning electron microscopy, transmission electron microscopy, nanoparticle tracking analysis, and LC–MS/MS. The inhibitory activity of PFEVs against receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation was investigated in RAW 264.7 cells. PFEVs significantly decreased the expression levels of genes and proteins related to osteoclast differentiation. PFEVs decreased RANK–RANKL binding. In a CIA mouse model, PFEVs treatment significantly reduced arthritis scores and collagen-specific immunoglobulins. PFEVs treatment also reduced pro-inflammatory cytokines and increased anti-inflammatory cytokines. The anti-inflammatory effects were confirmed by H&E staining, and PFEVs treatment inhibited osteoclastogenesis in the CIA mouse model. In conclusion, PFEVs inhibited osteoclast differentiation by inhibiting RANK–RANKL signaling, thereby decreasing the expression of osteoclast differentiation-related genes. PFEVs also improved collagen-induced arthritis by inhibiting inflammation and osteoclastogenesis.

Isolation and Characterization of Exosomes from Ocular Samples.

Exosomes are microsize vesicles secreted by nearly all cells to the extracellular space. The vesicles transport cell signaling and communicate with other cells. Ultracentrifugation is the standard method to isolate exosomes from culture media or body fluid. Without ultracentrifuge, exosomes can be precipitated by polyethylene glycol or separated by size exclusion chromatography. After isolation, nanoparticle tracking analysis can help to estimate the size and concentration of exosome samples. Transmission electron microscopy can directly show the size and morphology of exosomes. Moreover, the sample should be characterized by the expression of several exosome biomarker proteins. Exosomal contents such as proteins and miRNAs could be profiled using appropriate technologies.

Detection of renal tubular transporter proteins in canine urinary extracellular vesicles using liquid chromatography-tandem mass spectrometry.

Urinary extracellular vesicles (UEVs) are membranous particles that carry renal tubular transporter proteins. Here, we evaluate whether selected renal tubular transporter proteins can be detected in UEVs isolated from small volume (1-5 mL) canine urine samples of healthy dogs and canine patients with elevated circulating parathyroid hormone (PTH)/PTH-related peptide (PTHrp) concentrations, hypercortisolism, and primary hypoadrenocorticism using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The total creatinine content of each urine sample was calculated from urine volume and creatinine concentration. UEVs were isolated by size exclusion chromatography prior to quantification by nanoparticle tracking analysis and proteomic analysis by LC-MS/MS. Group comparisons were made using non-parametric statistics. Aquaporin-2 (AQP2) and the renal sodium/phosphate co-transporter (NPT2A) were detected in UEVs isolated from small volume samples of almost all healthy dogs but were not detected in most dogs with elevated circulating PTH/PTH related peptide (PTHrp) concentrations, hypercortisolism and primary hypoadrenocorticism. Total creatinine content of the urine sample was strongly positively correlated with the number of UEVs (rs = .84, P < .001); thus, total creatinine was used as a surrogate marker of UEV number. In healthy dogs, AQP2 and NPT2A were both detected in samples containing at least 1.7 × 109 UEVs or 24 μmol creatinine, however in non-healthy dogs, AQP2 and NPT2A were not detected in most samples containing up to 6.3 × 109 UEVs or 32 μmol creatinine.

Pancreatic β-cells package double C2-like domain beta protein into extracellular vesicles via tandem C2 domains.

Double C2-like domain beta (DOC2B) is a vesicle priming protein critical for glucose-stimulated insulin secretion in β-cells. Individuals with type 1 diabetes (T1D) have lower levels of DOC2B in their residual functional β-cell mass and platelets, a phenotype also observed in a mouse model of T1D. Thus, DOC2B levels could provide important information on β-cell dys(function). Our objective was to evaluate the DOC2B secretome of β-cells. In addition to soluble extracellular protein, we assessed DOC2B localized within membrane-delimited nanoparticles - extracellular vesicles (EVs). Moreover, in rat clonal β-cells, we probed domains required for DOC2B sorting into EVs. Using Single Extracellular VEsicle Nanoscopy, we quantified EVs derived from clonal β-cells (human EndoC-βH1, rat INS-1 832/13, and mouse MIN6); two other cell types known to regulate glucose homeostasis and functionally utilize DOC2B (skeletal muscle rat myotube L6-GLUT4myc and human neuronal-like SH-SY5Y cells); and human islets sourced from individuals with no diabetes (ND). EVs derived from ND human plasma, ND human islets, and cell lines were isolated with either size exclusion chromatography or differential centrifugation. Isolated EVs were comprehensively characterized using dotblots, transmission electron microscopy, nanoparticle tracking analysis, and immunoblotting. DOC2B was present within EVs derived from ND human plasma, ND human islets, and INS-1 832/13 β-cells. Compared to neuronal-like SH-SY5Y cells and L6-GLUT4myc myotubes, clonal β-cells (EndoC-βH1, INS-1 832/13, and MIN6) produced significantly more EVs. DOC2B levels in EVs (over whole cell lysates) were higher in INS-1 832/13 β-cells compared to L6-GLUT4myc myotubes; SH-SY5Y neuronal-like cells did not release appreciable DOC2B. Mechanistically, we show that DOC2B was localized to the EV lumen; the tandem C2 domains were sufficient to confer sorting to INS-1 832/13 β-cell EVs. Clonal β-cells and ND human islets produce abundant EVs. In cell culture, appreciable DOC2B can be packaged into EVs, and a small fraction is excreted as a soluble protein. While DOC2B-laden EVs and soluble protein are present in ND plasma, further studies will be necessary to determine if DOC2B originating from β-cells significantly contributes to the plasma secretome.

Comparative Analysis of Extracellular Vesicles from Cytotoxic CD8+ αβ T Cells and γδ T Cells.

Although belonging to different branches of the immune system, cytotoxic CD8+ αβ T cells and γδ T cells utilize common cytolytic effectors including FasL, granzymes, perforin and granulysin. The effector proteins are stored in different subsets of lysosome-related effector vesicles (LREVs) and released to the immunological synapse upon target cell encounter. Notably, in activated cells, LREVs and potentially other vesicles are continuously produced and released as extracellular vesicles (EVs). Presumably, EVs serve as mediators of intercellular communication in the local microenvironment or at distant sites. EVs of activated and expanded cytotoxic CD8+ αβ T cells or γδ T cells were enriched from culture supernatants by differential and ultracentrifugation and characterized by nanoparticle tracking analyses and Western blotting. For a comparative proteomic profiling, EV preparations from both cell types were isobaric labeled with tandem mass tags (TMT10plex) and subjected to mass spectrometry analysis. 686 proteins were quantified in EV preparations of cytotoxic CD8+ αβ T cells and γδ T cells. Both populations shared a major set of similarly abundant proteins, while much fewer proteins presented higher abundance levels in either CD8+ αβ T cells or γδ T cells. To our knowledge, we provide the first comparative analysis of EVs from cytotoxic CD8+ αβ T cells and γδ T cells.

Rivaroxaban, in combination with low-dose aspirin, is associated with a reduction in proinflammatory and prothrombotic circulating vesicle signatures in patients with cardiovascular disease

BackgroundDespite secondary prevention with aspirin, patients with stable cardiovascular disease (CVD) remain at elevated long-term risk of major adverse cardiovascular events. The Cardiovascular Outcomes in People Using Anticoagulant Strategies (COMPASS) double-blind, randomized clinical trial demonstrated that aspirin plus low-dose rivaroxaban (COMPASS regime) significantly decreased the incidence of major adverse cardiovascular events by 24% compared with aspirin alone. However, the mechanisms underlying these potential synergistic/nonantithrombotic effects remain elusive. Extracellular vesicles (EVs) are crucial messengers regulating a myriad of biological/pathological processes and are highly implicated in CVD. ObjectivesWe hypothesized that circulating EV profiles reflect the cardioprotective properties of the COMPASS regime. MethodsA cohort of stable CVD patients (N = 40) who participated in the COMPASS trial and were previously randomized to receive aspirin were prospectively recruited and assigned a revised regimen of open-label aspirin plus rivaroxaban. Blood samples were obtained at baseline (aspirin only) and 6-month follow-up. Plasma EV concentration, size, and origin were analyzed by nanoparticle tracking analysis and flow cytometry. EVs were enriched by ultracentrifugation for proteomic analysis. ResultsThe COMPASS regime fundamentally altered small (<200 nm) and large (200-1000 nm) EV concentration and size compared with aspirin alone. Crucially, levels of platelet-derived and myeloperoxidase-positive EVs became significantly decreased at follow-up. Comparative proteomic characterization further revealed a significant decrease in highly proinflammatory protein expression at follow-up. ConclusionThe observed changes in EV subpopulations, together with the differential protein expression profiles, suggest amelioration of an underlying proinflammatory and prothrombotic state upon dual therapy, which may be of clinical relevance toward understanding the fundamental mechanism underlying the reported superior cardiovascular outcomes associated with this antithrombotic regimen.

Exosomal let-7b-5p deriving from parietal epithelial cells attenuate renal fibrosis through suppression of TGFβR1 and ARID3a in obstructive kidney disease.

As renal progenitor cells, parietal epithelial cells (PECs) have demonstrated multilineage differentiation potential in response to kidney injury. However, the function of exosomes derived from PECs has not been extensively explored. Immunofluorescent staining of Claudin-1 was used to identify primary PECs isolated from mouse glomeruli. Transmission electron microscopy, nanoparticle tracking analysis, and western blotting were used to characterize the properties of PECs-derived exosomes (PEC-Exo). The therapeutic role of PEC-Exo in tubulointerstitial fibrosis was investigated in the unilateral ureteral obstruction (UUO) mouse model and TGF-β1-stimulated HK-2 cells. High-throughput miRNA sequencing was employed to profile PEC-Exo miRNAs. One of the most enriched miRNAs in PEC-Exo was knocked down by transfecting miRNA inhibitor, and then we investigated whether this candidate miRNA was involved in PEC-Exo-mediated tubular repair. The primary PECs expressed Claudin-1, PEC-Exo was homing to obstructed kidney, and TGF-β1 induced HK-2 cells. PEC-Exo significantly alleviated renal inflammation and ameliorated tubular fibrosis both invivo and invitro. Mechanistically, let-7b-5p, highly enriched in PEC-Exo, downregulated the protein levels of transforming growth factor beta receptor 1(TGFβR1) and AT-Rich Interaction Domain 3A(ARID3a) in tubular epithelial cells (TECs), leading to the inhibition of p21 and p27 to restoring cell cycle. Furthermore, administration of let-7b-5p agomir mitigated renal fibrosis invivo. Our findings demonstrated that PEC-derived exosomes significantly repressed the expression of TGFβR1 and ARID3a by delivering let-7b-5p, thereby alleviating renal fibrosis. This study provides novel insights into the role of PEC-Exo in the repair of kidney injury and new ideas for renal fibrosis intervention.

M1 macrophages deliver CASC19 via exosomes to inhibit the proliferation and migration of colon cancer cells.

Colorectal cancer (CRC) continues to be one of the leading causes of cancer-related death worldwide. Exosomes have been established to play an important role in intercellular communication and that long non-coding RNA (lncRNA) CASC19 is enriched within M1 macrophage-derived exosomes (M1-exo). However, the biological functions and underlying molecular mechanisms of exosomal CASC19 from macrophages on CRC remain unknown. Cell proliferation and migration were evaluated by MTS and transwell assays. The exosomes were characterized by western blot, nanoparticle tracking analysis (NTA) and electron microscope imaging. The expression levels of CASC19 and its putative target miR-410-3p were quantified by reverse-transcription polymerase chain reaction (RT-qPCR). The interaction between CASC19 and miR-410-3p was detected by the pull-down assay. We found that the non-contact inhibition of M1 macrophages on the proliferation of colon cancer cells is largely dependent on the CASC19 released from M1 exosomes. M1 exosomes successfully delivered CASC19 to colon cancer cells, exerting an inhibitory effect on cell proliferation and migration. The exosomes secreted by M1 cells with CASC19 knockdown showed less inhibition effect on cell proliferation and migration. Mechanically, CASC19 exerted an inhibitory effect on colon cancer cells by sponging miR-410-3p via tube morphogenesis and TGF-β signaling pathway. We first proved that CASC19 in M1 macrophages is delivered into colon cancer cells via exosomes, exerting an inhibitory effect on their proliferation and migration by sponging miR-410-3p. The study may provide mechanistic insights into the roles of lncRNAs in CRC progression and a potential therapeutic target for the treatment of CRC.