Differential Centrifugation Method Research Articles

#348 Neutrophil membrane-derived nanovesicles deliver IL-37 to repair renal endothelial cells in ischemia‒reperfusion injury

Abstract Background and Aims Renal ischemia‒reperfusion injury (IRI) is one of the most important causes of acute kidney injury (AKI). Interleukin (IL)-37 is a novel anti-inflammatory factor, but its application is still limited by its low stability and delivery efficiency. In recent years, engineered extracellular vesicles (EVs) have attracted substantial attention as an emerging drug delivery system. The aim of this study was to investigate the reparative effect of neutrophil membranes-derived nanovesicle (N-MV)-delivered IL-37 on renal IRI and its mechanism of action. Method The density gradient centrifugation method was used to extract peripheral blood neutrophils from rats, and the differential centrifugation method was used to extract neutrophil membranes. Cell membranes and IL-37 were then extruded with polycarbonate porous membranes with different pore sizes using a mini extruder to prepare IL-37 that was coated with neutrophil membrane (N-MV@IL-37). Subsequently, the physical and chemical properties and in vitro biological functions were identified. The in vivo distribution of N-MV@IL-37 as well as the renal function and inflammatory response in rats were further evaluated in a rat renal IRI model, and the targeting and anti-inflammatory mechanisms of N-MV@IL-37 were further explored. Results N-MV@IL-37 not only enhanced the stability of IL-37 but also targeted endothelial cells via P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophil membrane surfaces. In vitro experiments showed that N-MV@IL-37 inhibited endothelial cell apoptosis and inflammatory factor production and promoted endothelial cell viability and angiogenesis. In the rat renal IRI model, N-MV@IL-37 inhibited renal cell apoptosis and alleviated inflammatory responses, thereby improving renal function in IRI rats. Conclusion N-MVs provide an effective method of delivering IL-37, thereby alleviating the inflammatory response due to renal IRI and repairing renal endothelial cells; these vesicles could be a potential system for the treatment of renal IRI. Meanwhile, N-MVs also provided a drug delivery platform to provide a new strategy for the treatment of other IRI-like diseases.

Human plasma derived exosomes: Impact of active and passive drug loading approaches on drug delivery

The aim of the current study was to explore the potential of human plasma-derived exosomes as versatile carriers for drug delivery by employing various active and passive loading methods. Exosomes were isolated from human plasma using differential centrifugation and ultrafiltration method. Drug loading was achieved by employing sonication and freeze thaw methods, facilitating effective drug encapsulation within exosomes for delivery. Each approach was examined for its effectiveness, loading efficiency and ability to preserve membrane stability. Methotrexate (MTX), a weak acid model drug was loaded at a concentration of 2.2 µM to exosomes underwent characterization using various techniques such as particle size analysis, transmission electron microscopy and drug loading capacity. Human plasma derived exosomes showed a mean size of 162.15 ± 28.21 nm and zeta potential of −30.6 ± 0.71 mV. These exosomes were successfully loaded with MTX demonstrated a better drug encapsulation of 64.538 ± 1.54 % by freeze thaw method in comparison 55.515 ± 1.907 % by sonication. In-vitro drug release displayed 60 % loaded drug released within 72 h by freeze thaw method that was significantly different from that by sonication method i.e., 99 % within 72 h (p value 0.0045). Moreover, cell viability of exosomes loaded by freeze thaw method was significantly higher than that by sonication method (p value 0.0091) suggested that there was membrane disruption by sonication method. In conclusion, this study offers valuable insights into the potential of human plasma-derived exosomes loaded by freeze thaw method suggest as a promising carrier for improved drug loading and maintenance of exosomal membrane integrity.

Approaches for attaining clean bacterial fractions from complex environmental samples

Marine bacteria have been targeted by industry and pharmaceutics as genetic resources for highly active enzymes or novel lead compounds. Although numerous techniques have been introduced to isolate useful bacteria from the environment, we are still highly dependent on the conventional direct cultivation method to attain pure cultures. However, efficient bacterial isolation is hindered by several factors, including the presence of impurities. In this work, to demonstrate the significance of removing impurities and their impact on bacterial isolation, we employed two approaches: dielectrophoresis (DEP) and fluorescent D-amino acids (FDAA). We successfully attained clean bacterial fractions applicable for downstream processing using these approaches, uniquely designed to identify bacteria based on their characteristics and features. The diversity of bacteria attained by both approaches was investigated using 16S rRNA sequencing and compared to that attained by the standard differential centrifugation method. In addition, the viability of the isolates was also determined via direct cultivation. As a result, the separation of bacteria from impurities allowed for the identification of novel and useful bacteria unique to each approach. Successful cultivation also suggested that both approaches were applicable for attaining viable bacteria. In conclusion, removing impurities to attain clean bacterial fractions promotes the isolation of novel bacteria and thus could aid in the successful isolation of useful bacteria within complex environmental samples.

Abstract 5875: Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance

Abstract Background and Aim: Multidrug resistance (MDR) is an unresolved obstacle to cancer chemotherapy. It is usually caused by overexpression of drug efflux transporters including ABCG2. Exosomes are extracellular lipid vesicles released from all cells upon the fusion of multivesicular bodies with plasma membrane. Tumor-derived exosomes have been shown to mediate cell-to-cell transfer of oncogenic materials to promote drug resistance. Human antigen R (HuR) is an extensively studied RNA binding protein that regulates the stability, translation, and nucleus-to-cytoplasm shuttling of mRNAs. Numerous HuR-targeting mRNAs play crucial roles in tumorigenesis, invasion and metastasis. We have previously reported the induction of ABCG2 and MDR phenotype by HuR overexpression in colon cancer. HuR overexpression is linked with high-grade malignancy and poor prognosis in cancer patients. This study investigated the enrichment of HuR inducing microRNAs (miRNAs) in exosomes secreted from MDR colon cancer and its intracellular transfer to drive MDR. Method: Cancer exosome-mediated transfer of miRNAs from ABCG2-overexpressing resistant colon cancer cells S1M1-80 to a few sensitive colon cell lines was evaluated. Exosomes were isolated from S1M1-80 conditioned medium by differential centrifugation method. The uptake of fluorescence-labeled exosome from donor MDR cancer cells by recipient sensitive cells was visualized by fluorescence microscopy. MiRNA microarray was conducted to identify differentially expressed miRNAs in drug sensitive cells after incubation with S1M1-80-secreted exosomes. Western blot and qPCR analysis were conducted to measure HuR and ABCG2 expression. Result: Exosomes were isolated from resistant S1M1-80 cells, whose purity was confirmed by the presence of exosomal markers (Tsg101 and Alix) but absence of cellular marker (GRP78) by Western blot analysis. These exosomes reduced the apoptotic effect of SN-38 or 5-fluorouracil in sensitive colon cancer cell lines (HCT116, S1, and SW480) by inducing both HuR and ABCG2. A common upregulated miRNA (miR-155-5p) was identified by miRNA microarray in the sensitive cell lines after incubation with S1M1-80-derived exosomes. MiR-155-5p was found to induce HuR expression by promoting its translation. MiR-155-5p inhibitor was shown to reverse the upregulation of HuR in HCT116 cells after exosome incubation, which subsequently suppressed ABCG2 expression and the MDR phenotype. Furthermore, inhibition of exosome uptake by dynasore was found to prevent the induction of HuR/ABCG2, and re-sensitize the exosome-treated sensitive cells to SN38. Conclusion: MDR cells-derived exosomes may spread the drug resistance phenotype by transferring a miR-155-5p/HuR/ABCG2 regulatory machinery to sensitive cells. Inhibition of this exosomal transfer of regulatory materials may prevent the spread of MDR and sensitize cancer cells to chemotherapy. Citation Format: Kenneth K.W. To. Upregulation of the RNA binding protein HuR by cancer-secreted exosome to mediate multidrug resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5875.

Approaching Effective Differential Centrifugal Fractionation by Combining Image Analysis with Analytical Ultracentrifugation.

Centrifugation is one of the most commonly used methods for separation in biology and chemistry. However, effective fractionation is not always easy to obtain, as preparative centrifuge experiments are mostly conducted in an empirical way, even when it is guided by the quantitative results from analytical ultracentrifuge (AUC). Very few works have been performed to enhance the fractionation resolution of the differential centrifugation method in a swing-out rotor. This is primarily due to the absence of a characterization tool for sedimentation in the preparative centrifuge. In this study, we utilized image analysis to map the particle concentration distribution throughout the preparative centrifuge tube, revealing an unexpected and abnormal sedimentation process. By characterizing the sedimentation coefficient distributions of the fractionated product via AUC, we demonstrated that the overall sedimentation efficiency in a swing-out preparative centrifuge was significantly reduced. Furthermore, effective fractionation was confined to the intermediate phase of the entire sedimentation process. We propose that the mechanism here is a combination of the inverse Boycott effect and droplet sedimentation. The actual sedimentation process within a preparative centrifuge can be described by modifying the Lamm equation phenomenologically, which simply results in an effective sedimentation coefficient. Our work builds a foundation for determining the optimal preparative centrifugation conditions for various systems.

A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia

A new effective method for isolating single spermatozoa from surgically obtained material in azoospermia

Tumor-derived exosomal miR-1247-3p promotes angiogenesis in bladder cancer by targeting FOXO1

ABSTRACTTumor-derived exosomes are highly correlated with tumor progression and angiogenesis. This study was designed to probe the role of tumor-derived exosomal miR-1247-3p in mediating the angiogenesis in bladder cancer. Exosomes isolation from the culture medium of normal or bladder cancer cell lines was performed using a differential centrifugation method. miR-1247-3p expression in exosomes and cells was detected by quantitative real-time PCR (qRT-PCR). The effect of exosomes on the angiogenesis of human umbilical vein endothelial cells (HUVECs) was assessed using cell counting kit-8 (CCK-8), transwell and tube formation assays. The interaction between miR-1247-3p and forkhead box protein O1 (FOXO1) was studied using luciferase reporter and RNA pull down assays. Exosomes were successfully isolated from T24, UM-UC-3, and SV-HUC-1 cells, as confirmed by corresponding identifications. Functional experiments revealed that exosomes derived from T24 and UM-UC-3 cells significantly enhanced the abilities of proliferation, migration, angiogenesis, and vascular endothelial-derived growth factor (VEGF) secretion in HUVECs. miR-1247-3p was highly expressed in exosomes derived from T24 and UM-UC-3 cells, and exosomes derived from miR-1247-3p inhibitor-transfected cells reduced HUVEC viability, migration, tube formation, and VEGF level. FOXO1 was confirmed as a direct target of miR-1247-3p. Rescue assays suggested that the effect of miR-1247-3p inhibition on the viability, migration, and angiogenesis of HUVECs was partly abrogated by the knockdown of FOXO1. Our data suggest that miR-1247-3p is up-regulated in tumor-derived exosomes, thereby inhibiting FOXO1 expression and facilitating angiogenesis in bladder cancer.

The use of platelet-rich plasma in osteoarthritis - the current state of knowledge

Introduction and objective: Osteoarthritis (OA) is a chronic condition characterized by progressive damage to the joint cartilage and bone. Platelet-rich plasma (PRP) has gained increasing interest as a potential therapeutic approach for relieving symptoms and improving joint function. The aim of this article is to present the current knowledge regarding the PRP preparation method, mechanisms of action, and treatment outcomes in OA based on scientific literature and recent research. Materials and methods: A review of scientific literature available on Google Scholar, PubMed, and Via Medica journal database was conducted using relevant keywords. State of knowledge: Platelet-rich plasma is obtained from the patient's own blood using differential centrifugation method, resulting in a concentration of platelets (PLT) that is 300-700% higher compared to the baseline. PLTs release various biologically active substances, including growth factors, which stimulate tissue repair and regeneration processes. Numerous studies have shown that PRP administration in patients with osteoarthritis of the knee and hip joints leads to pain reduction and improved joint function. The therapeutic effect yields long-lasting results, lasting up to 12 months after the completion of the treatment. Conclusions: The use of platelet-rich plasma (PRP) in the treatment of osteoarthritis presents a promising alternative to conventional therapies. However, further research is needed to confirm the effectiveness of PRP on a larger sample size and assess the long-term effects of this therapy. Knowledge regarding dosage, treatment protocols, and the duration of therapy remains limited.

Improving the Size Distribution of Polymeric Oblates Fabricated by the Emulsion-in-Gel Deformation Method

The optimization of fabrication conditions for colloidal micron-sized oblates obtained by the deformation of an oil-in-hydrogel emulsion is reported. The influence of the type of emulsion stabilizer, ultrasonication parameters, and emulsion and gel mixing conditions was explored. The best conditions with which to obtain more uniform particles were using polyvinyl alcohol as an emulsion stabilizer mixed with the gelatine solution at 35 °C and slowly cooling to room temperature. Four fractionation methods were applied to oblates to improve their size uniformity. The iterative differential centrifugation method produced the best size polydispersity reduction.

Selenium Biofortification Enhanced miR167a Expression in Broccoli Extracellular Vesicles Inducing Apoptosis in Human Pancreatic Cancer Cells by Targeting IRS1.

Pancreatic adenocarcinoma (PAAD) presents an extremely high morbidity and mortality rate. Broccoli has excellent anti-cancer properties. However, the dosage and serious side effects still limit the application of broccoli and its derivatives for cancer therapy. Recently, extracellular vesicles (EVs) derived from plants are emerging as novel therapeutic agents. Thus, we conducted this study to determine the effectiveness of EVs isolated from Se-riched broccoli (Se-BDEVs) and conventional broccoli (cBDEVs) for the treatment of PAAD. In this study, we first isolated Se-BDEVs and cBDEVs by a differential centrifugation method, and characterized them by using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Then, miRNA-seq was combined with target genes prediction, and functional enrichment analysis to reveal the potential function of Se-BDEVs and cBDEVs. Finally, the functional verification was conducted in PANC-1 cells. Se-BDEVs and cBDEVs exhibited similar characteristics in size and morphology. Subsequent miRNA-seq revealed the expression of miRNAs in Se-BDEVs and cBDEVs. Using a combination of miRNA target prediction and KEGG functional analysis, we found miRNAs in Se-BDEVs and cBDEVs may play an important role in treating pancreatic cancer. Indeed, our in vitro study showed that Se-BDEVs had greater anti-PAAD potency than cBDEVs due to increased bna-miR167a_R-2 (miR167a) expression. Transfection with miR167a mimics significantly induced apoptosis of PANC-1 cells. Mechanistically, further bioinformatics analysis showed that IRS1, which is involved in the PI3K-AKT pathway, is the key target gene of miR167a. This study highlights the role of miR167a transported by Se-BDEVs which could be a new tool for counteracting tumorigenesis.

Identifying Redox-Sensitive Cysteine Residues in Mitochondria.

The mitochondrion is the primary energy generator of a cell and is a central player in cellular redox regulation. Mitochondrial reactive oxygen species (mtROS) are the natural byproducts of cellular respiration that are critical for the redox signaling events that regulate a cell's metabolism. These redox signaling pathways primarily rely on the reversible oxidation of the cysteine residues on mitochondrial proteins. Several key sites of this cysteine oxidation on mitochondrial proteins have been identified and shown to modulate downstream signaling pathways. To further our understanding of mitochondrial cysteine oxidation and to identify uncharacterized redox-sensitive cysteines, we coupled mitochondrial enrichment with redox proteomics. Briefly, differential centrifugation methods were used to enrich for mitochondria. These purified mitochondria were subjected to both exogenous and endogenous ROS treatments and analyzed by two redox proteomics methods. A competitive cysteine-reactive profiling strategy, termed isoTOP-ABPP, enabled the ranking of the cysteines by their redox sensitivity, due to a loss of reactivity induced by cysteine oxidation. A modified OxICAT method enabled a quantification of the percentage of reversible cysteine oxidation. Initially, we assessed the cysteine oxidation upon treatment with a range of exogenous hydrogen peroxide concentrations, which allowed us to differentiate the mitochondrial cysteines by their susceptibility to oxidation. We then analyzed the cysteine oxidation upon inducing reactive oxygen species generation via the inhibition of the electron transport chain. Together, these methods identified the mitochondrial cysteines that were sensitive to endogenous and exogenous ROS, including several previously known redox-regulated cysteines and uncharacterized cysteines on diverse mitochondrial proteins.

Alcohol and HIV-Derived Hepatocyte Apoptotic Bodies Induce Hepatic Stellate Cell Activation.

Simple SummaryDid you know that alcohol abuse among people living with HIV (PLWH) is twice as frequent as in the general population? This undoubtedly makes alcohol abuse in the context of HIV a severe public health issue, which is often attributed to several comorbidities including liver disease. In fact, 50% of mortality from HIV-related liver disease in the United States is attributed to alcohol. Previously, we showed that an alcohol metabolite, acetaldehyde, drives HIV accumulation in hepatocytes, which generates oxidative stress leading to hepatocyte death. When large vesicles (apoptotic bodies) from dead hepatocytes are internalized by hepatic stellate cells (HSC), it causes their profibrotic activation. In fact, our data revealed that upon internalization of HIV-containing hepatocyte apoptotic bodies, HSC generate reactive oxygen species (ROS), which induce fibrosis by activating JNK-ERK1/2 and JAK-STAT3 pathways. Therefore, a potential therapeutic regimen for reducing liver fibrosis among PLWH should include ROS-scavenging antioxidants.Recently, we found that both HIV and acetaldehyde, an alcohol metabolite, induce hepatocyte apoptosis, resulting in the release of large extracellular vesicles called apoptotic bodies (ABs). The engulfment of these hepatocyte ABs by hepatic stellate cells (HSC) leads to their profibrotic activation. This study aims to establish the mechanisms of HSC activation after engulfment of ABs from acetaldehyde and HIV-exposed hepatocytes (ABAGS+HIV). In vitro experiments were performed on Huh7.5-CYP (RLW) cells to generate hepatocyte ABs and LX2 cells were used as HSC. To generate ABs, RLW cells were pretreated for 24 h with acetaldehyde, then exposed overnight to HIV1ADA and to acetaldehyde for 96 h. Thereafter, ABs were isolated from cell suspension by a differential centrifugation method and incubated with LX2 cells (3:1 ratio) for profibrotic genes and protein analyses. We found that HSC internalized ABs via the tyrosine kinase receptor, Axl. While the HIV gag RNA/HIV proteins accumulated in ABs elicited no productive infection in LX2 and immune cells, they triggered ROS and IL6 generation, which, in turn, activated profibrotic genes via the JNK-ERK1/2 and JAK-STAT3 pathways. Similarly, ongoing profibrotic activation was observed in immunodeficient NSG mice fed ethanol and injected with HIV-derived RLW ABs. We conclude that HSC activation by hepatocyte ABAGS+HIV engulfment is mediated by ROS-dependent JNK-ERK1/2 and IL6 triggering of JAK-STAT3 pathways. This can partially explain the mechanisms of liver fibrosis development frequently observed among alcohol abusing PLWH.

Efficient purification method for CsPbX3 perovskite quantum dots

CsPbX3 perovskite quantum dots (QDs) have attracted increasing attention for the applications of lighting and display due to their excellent properties. Although the preparation of perovskite QDs has been widely developed, there is still not a universal and efficient method to purify perovskite QDs, which is due to the labile ionic characteristics of the perovskites. Here, a new purification method is proposed by using the addition of a new antisolvent-dimethicone oil (DMO). Compared to the two commonly used purification methods (direct centrifugation and centrifugation with ethyl acetate antisolvent), the sample purified by DMO addition shows the highest photoluminescence quantum yield (PLQY) and stability. Besides, the yield of the samples obtained from DMO addition is also the highest. This new purification method is also suitable for the products prepared by the room temperature method, where large amounts of QDs with high PLQY can be acquired. Furthermore, a simple differential centrifugation method is designed based on this anti-solvent. Size-selected perovskite QDs with narrow size distribution are obtained from this differential centrifugation method.

Exosomes from human induced pluripotent stem cells derived mesenchymal stem cells improved myocardial injury caused by severe acute pancreatitis through activating Akt/Nrf2/HO-1 axis

ABSTRACT Human induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) have been believed to be a promising alternative for the stem cell transplantation therapy. The exosomes (Exo) from iMSCs play an important role in several kinds of life activities. The role of exosomes from iMSCs in severe acute pancreatitis (SAP) induced myocardial injury (MI) has not been investigated. The Exo were isolated from iMSCs through differential centrifugation method. The SAP rat model was established with 5% sodium taurocholate injection into the distal end of the bilepancreatic duct. RT-PCR and western blotting were used to measure related gene expression. Masson trichrome and Sirius Red stainings were used to evaluate MI injury. Cardiac function was detected through cardiac ultrasound.Exo promoted cell viability through activating Akt/nuclear factor E2 related factors 2 (Nrf2)/heme oxygenase 1 (HO-1) signaling pathway in vitro. Exo improved MI induced by SAP through activating Akt/Nrf2/HO-1 signaling pathway. Exo improved cardiac function, and suppressed oxidative status in the SAP model. Exo increased the expression of von Willebrand Factor (vWF) and vascular endothelial growth factor (VEGF) through activating Nrf2/HO-1 signaling pathway. Our data indicated that the Exo from iMSCs could improve MI caused by SAP through activating Nrf2/HO-1 axis. These findings firstly unfold the potential application of Exo from iMSCs in treating MI induced by SAP. Abbreviations: LVEF: Left ventricular ejection fraction; LVFS: left ventricular fractional shorten; LVDd: left ventricular end-diastolic diameter; LVDs: left ventricular end-systolic diameter; MI: Myocardial infarction; MSCs: Mesenchymal stem cells; iPSCs: Human-induced pluripotent stem cells; SAP: Severe acute pancreatitis; iMSCs: iPSCs derived VEGF: MSCs; vascular endothelial growth factor; Nrf2: Nuclear factor erythroid 2-related factor; RT-PCR: Real-time polymerase chain reaction; HE: Hematoxylin-eosin; MODS: Multiple organ dysfunction syndrome; PI3K: Phosphatidylinositol 3-kinase; SOD: Superoxide dismutase; FBS: Fetal bovine serum; ECL: Enhanced chemiluminescence; IHC: Immunohistochemistry.

Multicenter Evaluation of Rapid BACpro® II for the Accurate Identification of Microorganisms Directly from Blood Cultures Using MALDI-TOF MS.

The identification of microorganisms directly from blood cultures using MALDI-TOF MS has been shown to be the most impacting application of this methodology. In this study, a novel commercial method was evaluated in four clinical microbiology laboratories. Positive blood culture samples (n = 801) were processed using a rapid BACpro® II kit and then compared with the routine gold standard. A subset of monomicrobial BCs (n = 560) were analyzed in parallel with a Sepsityper® Kit (Bruker Daltonics, Bremen, Germany) and compared with the rapid BACpro® II kit. In addition, this kit was also compared with two different in-house methods. Overall, 80.0% of the monomicrobial isolates (609/761; 95% CI 71.5–88.5) were correctly identified by the rapid BACpro® II kit at the species level (92.3% of the Gram negative and 72.4% of the Gram positive bacteria). The comparison with the Sepsityper® Kit showed that the rapid BACpro® II kit generated higher rates of correct species-level identification for all categories (p > 0.0001), except for yeasts identified with score values > 1.7. It also proved superior to the ammonium chloride method (p > 0.0001), but the differential centrifugation method allowed for higher rates of correct identification for Gram negative bacteria (p > 0.1). The percentage of accurate species-level identification of Gram positive bacteria was particularly noteworthy in comparison with other commercial and in-house methods.

Ultra-fast direct method for identifying microorganisms from BACTEC lytic/10 anaerobic/F flasks.

Background: Fast diagnosis of bloodstream infections remains the most important challenge for clinical microbiologists. The introduction of the mass-spectrometry represents a breakthrough, although several methods are already commonly used for the direct identification from positive blood cultures we present a faster method (ultra fast) for Lytic anaerobic flasks. Methods: We compare the ultra-fast (UF) method with the extensively employed differential centrifugation method (DC) and both to routine identification after 18-24h of incubation. UF and DC method correlation rates to the gold standard were calculated, and statistical significance was proved with the Z test. Results: UF performed better overall than DC, with this difference being statistically significant. This tendency was observed in every subanalysis.

Telocytes-derived extracellular vesicles alleviate aortic valve calcification by carrying miR-30b.

AimsCalcific 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 resultsTCs 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.ConclusionTC‐EVs played a protective role in aortic valve calcification via the miR‐30b/Runx2/Wnt/β‐catenin axis.

Microvesicles and exosomes released by amnion epithelial cells under oxidative stress cause inflammatory changes in uterine cells†.

Extracellular vesicles play a crucial role in feto-maternal communication and provide an important paracrine signaling mechanism in pregnancy. We hypothesized that fetal cells-derived exosomes and microvesicles (MVs) under oxidative stress (OS) carry unique cargo and traffic through feto-maternal interface, which cause inflammation in uterine cells associated with parturition. Exosomes and MVs, from primary amnion epithelial cell (AEC) culture media under normal or OS-induced conditions, were isolated by optimized differential centrifugation method followed by characterization for size (nanoparticle tracking analyzer), shape (transmission electron microscopy), and protein markers (western blot and immunofluorescence). Cargo and canonical pathways were identified by mass spectroscopy and ingenuity pathway analysis. Myometrial, decidual, and cervical cells were treated with 1 × 107 control/OS-derived exosomes/MVs. Pro-inflammatory cytokines were measured using a Luminex assay. Statistical significance was determined by paired T-test (P< 0.05). AEC produced cup-shaped exosomes of 90-150nm and circular MVs of 160-400nm. CD9, heat shock protein 70, and Nanog were detected in exosomes, whereas OCT-4, human leukocyte antigen G, and calnexin were found in MVs. MVs, but not exosomes, were stained for phosphatidylserine. The protein profiles for control versus OS-derived exosomes and MVs were significantly different. Several inflammatory pathways related to OS were upregulated that were distinct between exosomes and MVs. Both OS-derived exosomes and MVs significantly increased pro-inflammatory cytokines (granulocyte-macrophage colony-stimulating factor, interleukin 6 (IL-6), and IL-8) in maternal cells compared with control (P< 0.05). Our findings suggest that fetal-derived exosomes and MVs under OS exhibited distinct characteristics and a synergistic inflammatory role in uterine cells associated with the initiation of parturition.

Exosome-mediated transfer of CD44 from high-metastatic ovarian cancer cells promotes migration and invasion of low-metastatic ovarian cancer cells

ObjectiveTo investigate the detailed roles and mechanisms of tumor-derived exosomes in progression and metastasis of ovarian cancer in vitro.MethodsExosomes were isolated by differential centrifugation method; the morphology, size and biological markers of exosomes were separately defined by transmission electron microscopy, nanoS90 and Western blotting; Trans-well chambers assay was used to assess the ability of migration and invasion of recipient cells uptaking the exosomes from HO8910PM cells. The downstream molecule was screened by mass spectrometry.CD44 was identified by western blotting and the function of CD44 was identified by trans-well chambers assay and CCK8 assay.ResultsExosomes derived from HO8910PM cells could be transferred to HO8910 cells and promote cell migration and invasion in the recipient cells of ovarian cancer. And CD44 could be transferred to the HO8910 cells through exosomes from HO8910PM cells and influence the migration and invasion ability of HO8910 cells.ConclusionThe more aggressive subpopulation can transfer a metastatic phenotype to the less one via secreting exosomes within a heterogeneous tumor. CD44 may be a potential therapeutic approach for ovarian cancer.

Selective loss of microvesicles is a major issue of the differential centrifugation isolation protocols

Microvesicles (MVs) are large extracellular vesicles differing in size, cargo and composition that share a common mechanism of release from the cells through the direct outward budding of the plasma membrane. They are involved in a variety of physiological and pathological conditions and represent promising biomarkers for diseases. MV heterogeneity together with the lack of specific markers had strongly hampered the development of effective methods for MV isolation and differential centrifugation remains the most used method to purify MVs. In this study, we analysed the capacity of the differential centrifugation method to isolate MVs from cell-conditioned medium using flow cytometry and TEM/AFM microscopy. We found that the loss of MVs (general population and/or specific subpopulations) represents a major and underestimate drawback of the differential centrifugation protocol. We demonstrate that the choice of the appropriate rotor type (fixed-angle vs swinging-bucket) and the implementation of an additional washing procedure to the first low-speed centrifugation step of the protocol allow to overcome this problem increasing the total amount of isolated vesicles and avoiding the selective loss of MV subpopulations. These parameters/procedures should be routinely employed into optimized differential centrifugation protocols to ensure isolation of the high-quantity/quality MVs for the downstream analysis/applications.