Sequential Ultracentrifugation Research Articles

Human Microglia Extracellular Vesicles Derived from Different Microglia Cell Lines: Similarities and Differences.

Microglial cells are a component of the innate immune system in the brain that support cell-to-cell communication via secreted molecules and extracellular vesicles (EVs). EVs can be divided into two major populations: large (LEVs) and small (SEVs) EVs, carrying different mediators, such as proteins, lipids, and miRNAs. The microglia EVs cargo crucially reflects the status of parental cells and can lead to both beneficial and detrimental effects in many physiopathological states. Herein, a workflow for the extraction and characterization of SEVs and LEVs from human C20 and HMC3 microglia cell lines derived, respectively, from adult and embryonic microglia is reported. EVs were gathered from the culture media of the two cell lines by sequential ultracentrifugation steps and their biochemical and biophysical properties were analyzed by Western blot, transmission electron microscopy, and dynamic light scattering. Although the C20- and HMC3-derived EVs shared several common features, C20-derived EVs were slightly lower in number and more polydispersed. Interestingly, C20- but not HMC3-SEVs were able to interfere with the proliferation of U87 glioblastoma cells. This correlated with the different relative levels of eight miRNAs involved in neuroinflammation and tumor progression in the C20- and HMC3-derived EVs, which in turn reflected a different basal activation state of the two cell types. Our data fill a gap in the community of microglia EVs, in which the preparations from human cells have been poorly characterized so far. Furthermore, these results shed light on both the differences and similarities of EVs extracted from different human microglia cell models, underlining the need to better characterize the features and biological effects of EVs for therein useful and correct application.

Abstract 189: Evaluating the influence of Lactobacillus rhamnosus-derived extracellular vesicles on calcium deposition in early breast cancer

Abstract Microcalcifications are an important mammographic feature of ductal carcinoma in situ (DCIS) and are detected in 85-90% of abnormal mammograms. Calcium transport and calcium channels regulate microcalcifications and are essential for breast cancer proliferation. The breast duct intraductal lumen, which is the origin of ductal carcinomas, has been shown to be a non-sterile environment. We hypothesize that the intraductal microbiome influences calcium handling and mineral deposition in breast cancer. Bacteria have been known to enhance the formation of mineral deposits, including calcium salts, in various non mammalian microenvironments via secreted factors or extracellular vesicle mediated nucleation. We performed a clinical study on a cohort of 150 women who received a suspicious mammogram and were subjected to a follow up biopsy, and we identified differences in calcium handling microbiota between patients who harbored a malignant disease with respect to benign controls. To mechanistically study the role of microbiota in breast cancer calcium handling, we developed a 3D spheroid model of ductal carcinoma in situ using the BT-474 cell line. When exposed to high concentrations of calcium in the culture medium (from 1.75 mM to 2.05 mM), spheroids developed calcium mineral deposits in a calcium dose dependent manner; deposits were identified in internal regions of the spheroid, which correspond to hypoxic areas of DCIS in vivo. Extracellular calcium deposition correlated with cell survival in face of a toxic calcium level. Spheroids were cultured under a continuous flow of Lactobacillus rhamnosus LMS2-1-derived EVs over the course of two weeks. EVs were isolated by sequential ultracentrifugation; the 100K fraction was retained and characterized in terms of size, concentration, and protein content. EV treatment induced increased calcium export and mineral deposition in a dose dependent manner. Our findings support the hypothesis that L. rhamnosus-derived extracellular vesicles influence calcium regulation and mineral deposition in the hypoxic breast cancer milieu. More studies will be undertaken in the future to unveil molecular mechanisms that underlie this interaction, both at the cancer cell and the microbiota level. Citation Format: Ngoc Bao Vuong, Melany Alomia, Intisar Alruwaili, Fahad Alsaab, James L. Erickson, Weidong Zhou, Alessandra Luchini, Monique Van Hoek, Virginia Espina, Richard A. Hoefer, Lance Liotta. Evaluating the influence of Lactobacillus rhamnosus-derived extracellular vesicles on calcium deposition in early breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 189.

The Preliminary Exploration of What Role miRNAs Derived From Urinary Exosomes Play in Kidney Stone Formation

To identify dysregulated microRNAs (miRNAs) derived from urinary small Extracellular Vesicles (sEVs) and to explore the potential roles sEVs miRNAs play in nephrolithiasis. First morning voids were collected from participants. Urinary exosomes were isolated via sequential differential centrifugation and ultracentrifugation. Next-generation sequencing was conducted to analyze the transcriptome of urine sEVs from three Calcium oxalate (CaOx) stone patients and three age-/sex-matched healthy controls. Bioinformatics analysis was performed to explore how dysregulated miRNAs were associated with stone formation. The miRNA sequencing results of sEVs derived from epithelial cells (GSE110509) were compared with the dysregulated miRNAs of our study, and finally we found three common miRNAs. 54 dysregulated miRNAs were found between control and stone groups, among which 14 miRNAs were down-regulated and 40 miRNAs were up-regulated in stone groups compared to control groups. Gene ontology (GO) analysis indicated that dysregulated miRNAs were enriched in oxidative stress (Biological Process), focal adhesion (Cellular Component), and cell adhesion molecule binding (Molecular Function). Kyoto Encyclopedia of Genes and Genomes pathways analysis demonstrated that dysregulated miRNAs were common enriched in MAPK signaling pathway, focal adhesion and AGE-RAGE signaling pathway. The expression of miR-223-3p, miR-127-3p, and miR-214-5p were significantly different in either the sEVs derived from cells (GSE110509) or the sEVs derived from urine. QPCR confirmed that the expression of miR-223-3p was higher in urinary sEVs derived from CaOx stone patients. The expression of miR-223-3p is significantly higher in urinary sEVs derived from CaOx stone patients and may affect stone formation via regulating oxidative stress, focal adhesion and inflammation process.

Human Bone Marrow Stromal Cell Exosomes Ameliorate Periodontitis

Human bone marrow stromal cell (hBMSC)–derived exosomes are promising therapeutics for inflammatory diseases due to their unique microRNA (miRNA) and protein cargos. Periodontal diseases often present with chronicity and corresponding exuberant inflammation, which leads to loss of tooth support. In this study, we explored whether hBMSC exosomes can affect periodontitis progression. hBMSC exosomes were isolated from cell culture medium through sequential ultracentrifugation. miRNAs and proteins that were enriched in hBMSC exosomes were characterized by RNA sequencing and protein array, respectively. hBMSC exosomes significantly suppressed periodontal keystone pathogen Porphyromonas gingivalis–triggered inflammatory response in macrophages in vitro. Transcriptomic analysis suggested that exosomes exerted their effects through regulating cell metabolism, differentiation, and inflammation resolution. In vivo, weekly exosome injection into the gingival tissues reduced the tissue destruction and immune cell infiltration in rat ligature-induced periodontitis model. Collectively, these findings suggest that hBMSC-derived exosomes can potentially be used as a host modulation agent in the management of periodontitis.

Cellular prion protein in human plasma–derived extracellular vesicles promotes neurite outgrowth via the NMDA receptor–LRP1 receptor system

Exosomes and other extracellular vesicles (EVs) participate in cell–cell communication. Herein, we isolated EVs from human plasma and demonstrated that these EVs activate cell signaling and promote neurite outgrowth in PC-12 cells. Analysis of human plasma EVs purified by sequential ultracentrifugation using tandem mass spectrometry indicated the presence of multiple plasma proteins, including α2-macroglobulin, which is reported to regulate PC-12 cell physiology. We therefore further purified EVs by molecular exclusion or phosphatidylserine affinity chromatography, which reduced plasma protein contamination. EVs subjected to these additional purification methods exhibited unchanged activity in PC-12 cells, even though α2-macroglobulin was reduced to undetectable levels. Nonpathogenic cellular prion protein (PrPC) was carried by human plasma EVs and essential for the effects of EVs on PC-12 cells, as EV-induced cell signaling and neurite outgrowth were blocked by the PrPC-specific antibody, POM2. In addition, inhibitors of the N-methyl-d-aspartate (NMDA) receptor (NMDA-R) and low-density lipoprotein receptor–related protein-1 (LRP1) blocked the effects of plasma EVs on PC-12 cells, as did silencing of Lrp1 or the gene encoding the GluN1 NMDA-R subunit (Grin1). These results implicate the NMDA-R–LRP1 complex as the receptor system responsible for mediating the effects of EV-associated PrPC. Finally, EVs harvested from rat astrocytes carried PrPC and replicated the effects of human plasma EVs on PC-12 cell signaling. We conclude that interaction of EV-associated PrPC with the NMDA-R–LRP1 complex in target cells represents a novel mechanism by which EVs may participate in intercellular communication in the nervous system.

Isolation and immunogenicity of extracted outer membrane vesicles from Pseudomonas aeruginosa under antibiotics treatment conditions.

Background and Objectives:Different types of antibiotics have been indicated to enhance the secretion of OMVs from Pseudomonas aeruginosa. We aimed to investigate the effect of meropenem and amikacin antibiotics on inducing the secretion of OMVs and immunologic features in P. aeruginosa.Materials and Methods:The OMVs were prepared from P. aeruginosa under hypervesiculation condition (treatment with amikacin and meropenem), and extraction was carried out by the sequential ultracentrifugation. Physicochemical features of extracted OMVs were evaluated by electron microscopy and SDS-PAGE. To quantify antibody synthesis and function after immunization with OMV, we used ELISA, serum bactericidal activity, and opsonophagocytosis. Production of cytokines from splenocytes of immunized mice was measured with ELISA.Results:Specific-antibody IgG production, particularly IgG1 subclass, increased in mice primed with hypervesiculation-derived OMVs compared to normal condition-derived OMVs. Serum bactericidal activity and opsonophagocytosis of secreted antibody was enhanced in mice primed with hypervesiculation-derived OMVs. Investigation of cytokine production showed the upregulation of IL-8, IL-12, IL-17, and TNF-α and downregulation of IL-10.Conclusion:Based on our findings, OMVs production can be increased by treating P. aeruginosa with amikacin and meropenem antibiotics. Moreover, hypervesiculation-derived OMV scan possibly activate the humoral and cellular immune response more than normal OMVs.

Reduced cholesterol efflux capacity of HDL particles from Alzheimer's disease patients is not related to differences in lipoprotein profiles.

High density lipoproteins (HDL) are primarily known for their critical role in regulating cellular cholesterol concentrations via their capacity to induce cholesterol efflux from cells. HDL are also carriers of apolipoprotein E (ApoE), which is the strongest known genetic risk factor for AD. The main objective of this study was to determine the cholesterol efflux capacity of HDL particles isolated from plasma of APOE genotyped AD patients vs. age-matched cognitively normal controls, and to characterize plasma lipoprotein profiles using size exclusion chromatography. HDL particles were isolated from plasma samples of 10 AD patients (n=5 APOE3/E3, n=5 APOE3/E4) and 5 cognitively normal controls (n=5 APOE3/E3) using sequential ultracentrifugation followed by size exclusion chromatography. FPLC chromatograms were processed to adjust baseline values and calculate area under the curve (AUC) for LDL, HDL, and albumin using UNICORN 5.0 software. AUCs were used to estimate the relative distributions of fractions, including small and large HDL, and peak elution volume was used to estimate particle size most abundant in each fraction. Cholesterol efflux capacity of the isolated HDL particles was determined in PBMC-derived macrophages using a fluorescently labeled cholesterol assay kit. HDL from AD patients had dramatically reduced cholesterol efflux capacity compared with controls (36.0% ± 1.2% vs. 26.0% ± 1.2%, P<0.0001), with no difference between AD patients by APOE genotype (APOE3/E3, 25.9% ± 0.9%; APOE3/E4, 26.5% ±1.3%; P=0.21). There were no significant differences in the relative distributions or average particle sizes of HDL, LDL, albumin, or large and small HDL between patients and controls. These results suggest neither the amount nor size distribution of HDL particles is adequate to detect the main difference driving the diminished cholesterol efflux capacity of HDL particles from AD patients. The results of this study support previous findings of decreased cholesterol efflux capacity of HDL in AD patients, and underscore the necessity for additional analyses to characterize the compositional differences driving this diminished functional capacity and to further understand HDL's role in AD pathology.

Isolation of extra-cellular vesicles in the context of pancreatic adenocarcinomas: Addition of one stringent filtration step improves recovery of specific microRNAs.

microRNAs (miRNA) in extracellular vesicles (EVs) have been investigated as potential biomarkers for pancreatic ductal adenocarcinoma (PDAC). However, a mixed population of EVs is often obtained using conventional exosome isolation methods for biomarker development. EVs are derived from different cellular processes and present in various sizes, therefore miRNA expression among them is undoubtedly different. We developed a simple protocol utilizing sequential filtration and ultracentrifugation to separate PDAC EVs into three groups, one with an average diameter of more than 220 nm, named operational 3 (OP3); one with average diameters between 100–220 nm, named operational 2 (OP2); and another with average diameters around 100 nm, named operational 1 (OP1)). EVs were isolated from conditioned cell culture media and plasma of human PDAC xenograft mice and early stage PDAC patients, and verified by nanoparticle tracking, western blot, and electronic microscopy. We demonstrate that exosome specific markers are only enriched in the OP1 group. qRT-PCR analysis of miRNA expression in EVs from PDAC cells revealed that expression of miR-196a and miR-1246, two previously identified miRNAs highly enriched in PDAC cell-derived exosomes, is significantly elevated in the OP1 group relative to the other EV groups. This was confirmed using plasma EVs from PDAC xenograft mice and patients with localized PDAC. Our results indicate that OP1 can be utilized for the identification of circulating EV miRNA signatures as potential biomarkers for PDAC.

The anti-inflammatory effects of equine bone marrow stem cell-derived extracellular vesicles on autologous chondrocytes.

BackgroundOsteoarthritis (OA) in the horse is an economic and welfare issue and there are no current disease modifying drugs available. Stem cells have been suggested as a therapeutic intervention for OA, originally on the basis of their regenerative capacity. However, it is hypothesised that mesenchymal stem cells (MSC) exert their effects via paracrine factors including the production of extracellular vesicles that can themselves recapitulate the MSC effects in the joint.ObjectivesTo isolate extracellular vesicles from bone marrow MSC and investigate their anti‐inflammatory effects on chondrocytes.Study designAn in vitro assessment of the effect of direct culturing extracellular vesicles on artificially inflamed chondrocytes.MethodsExtracellular vesicles were isolated from bone marrow MSC using differential sequential ultracentrifugation. Vesicles were characterised using electron microscopy, nanoparticle tracing analysis and protein analysis. Vesicle internalisation was carried out via vesicles being pre‐stained and co‐cultured with equine chondrocytes before analysis using confocal microscopy. The effects of vesicles on artificially inflamed chondrocytes was examined using quantitative PCR.ResultsTo the best of the authors' knowledge, this is the first study to isolate and characterise extracellular vesicles from equine bone MSC. Vesicles were taken up by autologous chondrocytes and had anti‐inflammatory effects on gene expression following chondrocyte exposure to tumour necrosis factor α and Interleukin 1β.Main limitationsOnly three independent biological repeats were performed and the work was done in vitro.ConclusionExtracellular vesicles can be isolated from equine bone marrow MSC; they may be taken up by chondrocytes and have an anti‐inflammatory action.

Surface Marker Expression in Small and Medium/Large Mesenchymal Stromal Cell-Derived Extracellular Vesicles in Naive or Apoptotic Condition Using Orthogonal Techniques.

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a promising resource for regenerative medicine. Small MSC-EVs represent the active EV fraction. A bulk analysis was applied to characterise MSC-EVs’ identity and purity, with the assessment of single EV morphology, size and integrity using electron microscopy. We applied different methods to quantitatively analyse the size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC-EVs obtained using sequential ultracentrifugation. Bone marrow, adipose tissue and umbilical cord MSC-EVs were compared in naive and apoptotic conditions. As detected by electron microscopy, the 100k EV size < 100 nm was confirmed by super-resolution microscopy and ExoView. Single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanins. ExoView allowed a comparative screening of single MSC-EV tetraspanin and mesenchymal markers. A semiquantitative bead-based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC-EVs. Apoptotic MSC-EVs were released in higher numbers, without significant differences in the naive fractions in surface marker expression. These results show a consistent profile of MSC-EV fractions among the different sources and a safer profile of the 100k MSC-EV population for clinical application. Our study identified suitable applications for EV analytical techniques.

Cytokine Profiling in Low- and High-Density Small Extracellular Vesicles from Epidermoid Carcinoma Cells

Exosomes or small extracellular vesicles (sEVs) are membrane-bound nanoparticles that carry various macromolecules and act as autocrine and paracrine signaling messengers. In this study, sEVs from epidermoid carcinoma cells influenced by membrane presentation of the glycoprotein desmoglein 2 and its palmitoylation state were investigated. In this study, sEVs were isolated by sequential ultracentrifugation followed by iodixanol density gradient separation. They were then subjected to multiplex profiling of cytokines associated with the surface of intact sEVs. The results revealed a previously undescribed active sorting of cytokines onto the surface of low-density and high-density sEV subpopulations. Specifically, an altered surface presentation of desmoglein 2 decreased FGF-2 and VEGF in low-density sEVs. In addition, in response to desmoglein 2, IL-8 and RANTES were increased in low-density sEVs but only slightly decreased in high-density sEVs. Finally, IL-6 and G-CSF were increased dramatically in high-density sEVs. This comprehensive analysis of the cytokine production profile by squamous cell carcinoma‒derived sEVs highlights their contribution to immune evasion, pro-oncogenic and proangiogenic activity, and the potential to identify diagnostic disease biomarkers.

LB719 Cytokine profiling in low- and high-density small extracellular vesicles from epidermoid carcinoma cells

Exosomes or small extracellular vesicles (sEVs) are membrane-bound, nanoparticles carrying various macromolecules, acting as autocrine and paracrine signaling messengers. Here, sEVs from epidermoid carcinoma cells influenced by membrane presentation of the glycoprotein desmoglein 2 and its palmitoylation state were investigated. sEVs were isolated by sequential ultracentrifugation followed by iodixanol density gradient separation and subjected to multiplex cytokine profiling. Subpopulations of different densities showed active sorting of surface-associated cytokines, chemokines and growth factors.

Isolation of HDL by sequential flotation ultracentrifugation followed by size exclusion chromatography reveals size-based enrichment of HDL-associated proteins

High-density lipoprotein (HDL) particles have multiple beneficial and cardioprotective roles, yet our understanding of their full structural and functional repertoire is limited due to challenges in separating HDL particles from contaminating plasma proteins and other lipid-carrying particles that overlap HDL in size and/or density. Here we describe a method for isolating HDL particles using a combination of sequential flotation density ultracentrifugation and fast protein liquid chromatography with a size exclusion column. Purity was visualized by polyacrylamide gel electrophoresis and verified by proteomics, while size and structural integrity were confirmed by transmission electron microscopy. This HDL isolation method can be used to isolate a high yield of purified HDL from a low starting plasma volume for functional analyses. This method also enables investigators to select their specific HDL fraction of interest: from the least inclusive but highest purity HDL fraction eluting in the middle of the HDL peak, to pooling all of the fractions to capture the breadth of HDL particles in the original plasma sample. We show that certain proteins such as lecithin cholesterol acyltransferase (LCAT), phospholipid transfer protein (PLTP), and clusterin (CLUS) are enriched in large HDL particles whereas proteins such as alpha-2HS-glycoprotein (A2HSG), alpha-1 antitrypsin (A1AT), and vitamin D binding protein (VDBP) are enriched or found exclusively in small HDL particles.

Compositional and functional properties of high-density lipoproteins in relation to coronary in-stent restenosis.

In-stent restenosis (ISR) is an unfavorable outcome that occurs in patients after coronary stenting. Use of drugs such as statins as well as drug-eluting stents has only been partially effective in reducing the rate of ISR. Since low high-density lipoprotein cholesterol (HDL-C) concentration is a pivotal cardiovascular disease risk factor, this study aimed to evaluate the compositional and functional alterations of HDL in individuals with ISR. This case-control study included 21 ISR, 26 non-ISR (NISR), 16 angiography-negative, and 18 healthy subjects. Serum HDL2 (d: 1.063-1.125 g/ml) and HDL3 (d: 1.125-1.210 g/ml) subfractions were extracted from each subject using sequential ultracentrifugation. The capacity of HDL to efflux cellular cholesterol from lipid-loaded macrophages as well as to take up free cholesterol (FC) from triglyceride-rich lipoproteins (TGRLs) during lipolysis was assessed. No difference was found in the HDL2 and HDL3 content of free cholesterol and total protein among the groups. The NISR group showed lower triglyceride content in HDL2 and higher phospholipid content in HDL3 relative to healthy subjects. Strong positive correlations were found between the cholesterol efflux capacity (CEC) of HDL2 and its phospholipid content in the healthy (r = 0.50), angiography-negative (r = 0.55) and ISR (r = 0.52) groups. The capacity of apolipoprotein B (apoB)-depleted serum to take up free cholesterol was not different among the groups. Despite some compositional alterations, the capacity of HDL to efflux cholesterol from lipid-loaded macrophages as well as to take up free cholesterol from TGRLs during lipolysis was not associated with ISR in this study.

Maternal and Child Supplementation With Small-Quantity Lipid-Based Nutrient Supplements Increases Child HDL Cholesterol Efflux Capacity

ObjectivesThe purpose of this secondary outcome analysis is to investigate whether small-quantity lipid-based nutrient supplements (SQ-LNS) alters lipid, protein or glycan composition, or the cholesterol efflux capacity (CEC), of high-density lipoprotein (HDL) particles in children in the International Lipid-Based Nutrient Supplements (iLiNS) DYAD trial in Ghana. MethodsPlasma samples were obtained from a subcohort of 80 children at 18 months of age from the iLiNS-DYAD-Ghana trial. Mothers were randomized to either iron and folic acid (IFA) in pregnancy and 200 mg/d calcium for 6 months postpartum or SQ-LNS (pregnancy and 6 months postpartum). Children in the SQ-LNS group received SQ-LNS from 6 to 18 months while children in the IFA group did not receive supplements. HDL was isolated from plasma by sequential ultracentrifugation followed by size-exclusion chromatography. Assay of cholesterol efflux was performed in vitro, and glycoproteomic and lipidomic composition were analyzed by mass spectrometry. The primary analysis was a comparison of the effects of intervention groups on HDL lipidome, proteome, and CEC. In the exploratory analysis, we compared the enrichment of glycopeptides in measured HDL-associated proteins between groups. ResultsMean (±SD) HDL CEC was higher among children in the SQ-LNS vs. IFA group (20.9 ± 4.1% vs. 19.4 ± 3.3%; one-tailed p = 0.038). We found no differences in HDL lipidomic or proteomic composition between groups. ConclusionsPrenatal and postnatal SQ-LNS may improve the CEC of child HDL particles. These improvements may have a potential impact on child health outcomes. Funding SourcesSupported by Bill & Melinda Gates Foundation grant to the University of California, Davis.

Cellular Uptake Heterogeneity of Small Extracellular Vesicles

Exosomes, also known as small extracellular vesicles (sEV), represent a subpopulation of EVs of 30-200nm in diameter that are contained into multivesicular bodies and are released by cells via exocytosis. There has been increased interest to use sEVs for therapeutic purposes due to their immune-tolerance, potential for targeted delivery and increased half-life in circulation, compare to other nanoparticles. However, due to the heterogeneity of sEVs as well as the different phenotypic features of recipient cells, the uptake mechanisms can be highly variable and selective. Studies from our lab and others show that, in vitro, not all the cells in a given cell population take up vesicles with similar kinetics and avidity. Therefore, understanding the uptake of sEVs by cells in different physiologic states (eg, quiescent vs. proliferative) as well as the intracellular fate of sEVs is key for their use as therapeutic tools. As proof of concept approaches, we used the interaction of sEVs from various endothelial cells with PC3-ML prostate cancer cells, a highly metastatic human cancer cell line. Using imaging flow cytometry and super-resolution microscopy, we determined the uptake of sEVs by a heterogeneous population of prostate cancer cells. sEVs were isolated by sequential ultracentrifugation, labeled with lipophilic dyes (DiD or DiO) and following incubation with recipient cells, nuclei were stained using DAPI or Hoechst dyes. Cells incubated with sEVs overnight showed a highly significant negative correlation between nuclear stain fluorescence and the internalization of labeled sEVs. Using two different nuclear dyes, we found concordant data (DAPI r = -.067 p < 0.0001 and Hoechst r = -.119, p < 0.0001).. This result was independently confirmed for DAPI nuclear staining using fluorescence microscopy (r = -.1352 p = 0.0004). We hypothesized that the negative correlation between nuclear staining and internalization may be cell cycle-dependent. Indeed, a binary switch was described from endocytosis being “on” in interphase to “off” in mitosis as cells traverse the G2/M checkpoint. Using clathrin-dependent endocytosis inhibitor dynasore, we found that >80% of the EVs uptake is via endocytosis. Internalization of sEVs was time-dependent and showed the same negative correlation with nuclear staining at each of the experimental time points (0,5, 1, 2, 4 and 24 hours). We also found that following incubation of cells with sEVs for up to 4hrs, co-localization with the cellular lysosomal compartment was <5%, suggesting that the majority of the internalized EVs escape lysosomal degradation following internalization. Further understanding of the uptake mechanisms of sEVs are key to set the premises for their use as therapeutic delivery systems in various diseases.

Экзосомы плазмы крови при наследственных формах болезни Паркинсона

Parkinson's disease (PD) is the second most common neurodegenerative disorder. Alpha-synuclein misfolding and aggregation resulting in neurototoxicity is a hallmark of PD. The prion properties of alpha-synuclein are still under discussion. Exosomes (extrcellular vesicles 40-100 nm in size) can play a key role in the transport of pathogenic forms of alpha-synuclein. The most frequent inherited forms of the disease are PD associated with mutation in the leucine-rich repeat kinase 2 (LRRK2-PD) and glucocerebrosidase (GBA-PD) genes. The aim of our work is to evaluate the concentration and size of exosomes derived from blood plasma of patients with GBA-PD, asymptomatic GBA mutation carriers, and the effect of GBA and LRRK2 mutations on alpha-synuclein level in exosomes derived from peripheral blood plasma. Plasma extracellular vesicles were isolated via chemical precipitation and sequential ultracentrifugation and characterized by transmission electron microscopy, nanoparticle tracking analysis (NTA), and flow cytometry. Total alpha-synuclein level in plasma exosomes was estimated by enzyme-linked immunosorbent assay. Patients with sporadic PD, PD with dementia, patients with inherited PD (GBA-PD, LRRK2-PD), and GBA mutation carriers were included in the study. The concentration on plasma exosomes was higher in GBA-PD patients that in sporadic PD patients, asymptomatic carriers of mutations on GBA gene, and control (p = 0.004, 0.019 and 0.0001 respectively). The size of plasma exosomes was higher in GBA-PD patients compared to asymptomatic carriers of GBA mutations and control (p = 0.009 and 0.0001, respectively). No significant difference was found for exosomal alpha-synuclein levels in the studied groups. Our results allowed us to suggest that a decrease in GBA activity may affect the pool of plasma exosomes, and mutations in the LRRK2 and GBA genes do not influence the level of plasma exosomal alpha-synuclein.

LncRNA Quantification from Extracellular Vesicles Isolated from Blood Plasma or Conditioned Media.

During the last years, the study of extracellular vesicles (EVs) and its cargo has gained interest in the scientific media. EVs have been found in all biofluids and it is postulated that all cells are capable to secrete a wide variety of these vesicles, which play a key role in different cell-to-cell communication processes as well as in the microenvironment modulation. In the EV cargo, DNA, protein, and RNA molecules can be found, including long noncoding RNAs (lncRNAs). Several authors consider the study of EV lncRNAs an ideal source of biomarkers due to the easy sampling of EVs in different biofluids and the high specificity of the lncRNA expression pattern.In the present chapter, a detailed explanation of the EV isolation workflow followed by RNA isolation and lncRNA gene expression study is provided for two sample sources: blood plasma and cell culture conditioned media. EVs from both plasma samples and cell cultured media are isolated using sequential ultracentrifugation method (UC), which has been reported as one of the best methods available to date in terms of purity. UC is followed by RNA extraction based on the combination of phenol/guanidine-based lysis of samples with silica-membrane-based purification of total RNA. LncRNA quantification is performed by qRT-PCR. This chapter includes detailed discussion on lncRNA quantification using hydrolysis probes, recommended housekeeping genes and evaluation of methods for comparing lncRNA levels between EVs and its parental cells. In summary, we describe here the main steps for a successful isolation of the EVs-lncRNA cargo, paying attention to how overcome the different challenges found in the experimental procedure and in the data analysis of lncRNA expression from this source.

HBMSC-Derived Extracellular Vesicles Attenuate IL-1β-Induced Catabolic Effects on OA-Chondrocytes by Regulating Pro-inflammatory Signaling Pathways

Background: Human bone marrow-derived mesenchymal stromal cells (hBMSCs) provide a promising therapeutic approach in the cell-based therapy of osteoarthritis (OA). However, several disadvantages evolved recently, including immune responses of the host and regulatory hurdles, making it necessary to search for alternative treatment options. Extracellular vesicles (EVs) are released by multiple cell types and tissues into the extracellular microenvironment, acting as message carriers during intercellular communication. Here, we investigate putative protective effects of hBMSC-derived EVs as a cell-free approach, on IL-1β-stimulated chondrocytes obtained from OA-patients.Methods: EVs were harvested from the cell culture supernatant of hBMSCs by a sequential ultracentrifugation process. Western blot, scanning electron microscopy (SEM), and nanoparticle tracking analysis (NTA) were performed to characterize the purified particles as EVs. Intracellular incorporation of EVs, derived from PHK26-labeled hBMSCs, was tested by adding the labeled EVs to human OA chondrocytes (OA-CH), followed by fluorescence microscopy. Chondrocytes were pre-stimulated with IL-1β for 24 h, followed by EVs treatment for 24 h. Subsequently, proliferation, apoptosis, and migration (wound healing) were analyzed via BrdU assay, caspase 3/7 assay, and scratch assay, respectively. With qRT-PCR, the relative expression level of anabolic and catabolic genes was determined. Furthermore, immunofluorescence microscopy and western blot were performed to evaluate the protein expression and phosphorylation levels of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB as components of pro-inflammatory signaling pathways in OA-CH.Results: EVs from hBMSCs (hBMSC-EVs) promote proliferation and reduce apoptosis of OA-CH and IL-1β-stimulated OA-CH. Moreover, hBMSC-EVs attenuate IL-1β-induced reduction of chondrocyte migration. Furthermore, hBMSC-EVs increase gene expression of PRG4, BCL2, and ACAN (aggrecan) and decrease gene expression of MMP13, ALPL, and IL1ß in OA-CH. Notably, COL2A1, SOX9, BCL2, ACAN, and COMP gene expression levels were significantly increased in IL-1β+ EV groups compared with those IL-1β groups without EVs, whereas the gene expression levels of COLX, IL1B, MMP13, and ALPL were significantly decreased in IL-1β+ EV groups compared to IL-1β groups without EVs. In addition, the phosphorylation status of Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling molecules, induced by IL-1β, is prevented by hBMSC- EVs.Conclusion: EVs derived from hBMSCs alleviated IL-1β-induced catabolic effects on OA-CH via promoting proliferation and migration and reducing apoptosis, probably via downregulation of IL-1ß-activated pro-inflammatory Erk1/2, PI3K/Akt, p38, TAK1, and NF-κB signaling pathways. EVs released from BMSCs may be considered as promising cell-free intervention strategy in cartilage regenerative medicine, avoiding several adverse effects of cell-based regenerative approaches.

In pregnancy, maternal HDL is specifically enriched in, and carries the highest proportion of, DHA in plasma

Arachidonic acid (AA) and docosahexaenoic acid (DHA) are important for neurological development. The aim was to determine the distribution and relative enrichment of AA and DHA among lipoprotein fractions prior to pregnancy, throughout gestation and in the post-partum period. Our hypothesis was that in pregnancy, in contrast to the non-pregnant state, AA and DHA are carried in highest concentration in the very low density lipoprotein (VLDL) fraction secondary to increased gestational liver triglyceride secretion. Two independent prospective, observational cohort studies carried out in Glasgow were combined; one early in pregnancy and one later in pregnancy with post-partum follow up. Across the pregnancy timeline plasma lipoproteins were isolated using sequential ultracentrifugation and lipoprotein fatty acids were extracted and analysed by gas chromatography. High density lipoprotein (HDL) had the highest concentration of AA and DHA compared to other lipoproteins. HDL became progressively enriched in the proportion of triglycerides at 16 weeks of gestation, which peaked at 35 weeks and returned to baseline at 13 weeks postpartum. HDL DHA per HDL-cholesterol and HDL DHA per apoA-I became progressively enriched at 16 weeks of gestation, peaked at 25 weeks and returned to baseline at 13 weeks postpartum, whereas HDL AA (per HDL-C or HDL-apoA-I) did not differ. DHA is carried primarily in HDL rather than VLDL. HDL has anti-oxidant properties that might afford DHA protection against oxidation.