Extracellular Vesicles Content Research Articles

Dynamic changes in extracellular vesicle-associated miRNAs elicited by ultrasound in inflammatory bowel disease patients

Blood-based biomarkers that reliably indicate disease activity in the intestinal tract are an important unmet need in the management of patients with IBD. Extracellular vesicles (EVs) are cell-derived membranous microparticles, which reflect the cellular and functional state of their site of site of origin. As ultrasound waves may lead to molecular shifts of EV contents, we hypothesized that application of ultrasound waves on inflamed intestinal tissue in IBD may amplify the inflammation-specific molecular shifts in EVs like altered EV-miRNA expression, which in turn can be detected in the peripheral blood. 26 patients with IBD were included in the prospective clinical study. Serum samples were collected before and 30 min after diagnostic transabdominal ultrasound. Differential miRNA expression was analyzed by sequencing. Candidate inducible EV-miRNAs were functionally assessed in vitro by transfection of miRNA mimics and qPCR of predicted target genes. Serum EV-miRNA concentration at baseline correlated with disease severity, as determined by clinical activity scores and sonographic findings. Three miRNAs (miR-942-5p, mir-5588, mir-3195) were significantly induced by sonography. Among the significantly regulated EV-miRNAs, miR-942-5p was strongly induced in higher grade intestinal inflammation and correlated with clinical activity in Crohn’s disease. Prediction of target regulation and transfection of miRNA mimics inferred a role of this EV-miRNA in regulating barrier function in inflammation. Induction of mir-5588 and mir-3195 did not correlate with inflammation grade. This proof-of-concept trial highlights the principle of induced molecular shifts in EVs from inflamed tissue through transabdominal ultrasound. These inducible EVs and their molecular cargo like miRNA could become novel biomarkers for intestinal inflammation in IBD.

In Situ Labeling of the Aqueous Compartment of Extracellular Vesicles with Luminescent Gold Nanoclusters.

Extracellular vesicles (EVs) are well-known membrane-limited particles secreted by both healthy and cancerous cells. They are considered as biomarkers for early cancer diagnosis and are involved in many pathologies and physiological pathways. They could serve as diagnostic tools in liquid biopsies, as therapeutics in regenerative medicine, or as drug delivery vehicles. Our aim is here to encapsulate luminescent nanoprobes in the aqueous compartment of human EVs extracted from reproductive fluids. The analysis and labeling of the EVs content with easily detectable luminescent nanoparticles could enable a powerful tool for early diagnosis of specific diseases and also for the design of new therapeutics. In this view, gold nanoclusters (AuNCs) appear as an attractive alternative as nontoxic fluorophore probes because of their luminescence properties, large window of fluorescence lifetimes (1 ns-1 μs), ultrasmall size (<2 nm), good biocompatibility, and specific ability as X-ray photosensitizers. Here, we investigated an attractive method that uses fusogenic liposomes to deliver gold nanoclusters into EVs. This approach guarantees the preservation of the EVs membrane without any breakage, thus maintaining compartmental integrity. Different lipid compositions of liposomes preloaded with AuNCs were selected to interact electrostatically with human EVs and compared in terms of fusion efficiency. The mixture of liposomes and EVs results in membrane mixing as demonstrated by FRET experiments and fusion revealed by flux cytometry and cryo-TEM. The resulting fused EVs exhibit typical fluorescence of the AuNCs together with an increased size in agreement with fusion. Moreover, the fusion events in mixtures of EVs and AuNCs preloaded liposomes were analyzed by using cryo-electron microscopy. Finally, the ratio of released AuNCs during the fusion between the fusogenic liposomes and the EVs was estimated to be less than 20 mol % by Au titration using ICP spectroscopy.

Impact of FADS genotype on polyunsaturated fatty acid content in human milk extracellular vesicles: A genetic association study.

Extracellular vesicles in human milk are critical in supporting newborn growth and development. Bioavailability of dietary extracellular vesicles may depend on the composition of membrane lipids. Single-nucleotide polymorphisms (SNPs) in the fatty acid desaturase gene cluster impact the content of long-chain polyunsaturated fatty acids in human milk phospholipids. This study investigated the relation between variation in FADS1 and FADS2 with the content of polyunsaturated fatty acids in extracellular vesicles from human milk. Milk was obtained from a cohort of mothers (N = 70) at 2-4 weeks of lactation. SNPs in the FADS gene locus were determined using pyrosequencing for rs174546 in FADS1 and rs174575 in FADS2. Quantitative lipidomic analysis of polyunsaturated fatty acids in human milk and extracellular vesicles from human milk was completed by gas chromatography-mass spectrometry. The rs174546 and rs174575 genotypes were independent predictors of the arachidonic acid content in extracellular vesicles. The rs174546 genotype also predicted eicosapentaenoic acid and docosahexaenoic acid in extracellular vesicles. The reduced content of long-chain polyunsaturated fatty acids in extracellular vesicles in human milk may be due to lower fatty acid desaturase activity in mothers who are carriers of the A allele in rs174546 or the G allele in rs174575. The polyunsaturated fatty acid composition of milk extracellular vesicles is predicted by the FADS genotype. These findings yield novel insights regarding extracellular vesicle content and composition that can inform the design of future research to explore how lipid metabolites impact the bioavailability of human milk extracellular vesicles.

Extracellular vesicles as potential diagnostic markers for kidney allograft rejection.

Kidney transplantation is a highly effective treatment for end-stage kidney disease. However, allograft rejection remains a significant clinical challenge in kidney transplant patients. Although kidney allograft biopsy is the gold-standard diagnostic method, it is an invasive procedure. Since the current monitoring methods, including screening of serum creatinine and urinary protein, are not of sufficient sensitivity, there is a need for effective post-transplant monitoring to detect allograft rejection at an early stage. Extracellular vesicles are vesicles with a lipid bilayer that originate from different cell types in pathological and physiological conditions. The content of extracellular vesicles reflects the status of cells at the time of their production. This review comprehensively summarizes clinical, in vivo, and in vitro reports that highlight the potential of extracellular vesicles as diagnostic biomarkers for kidney allograft rejection. Clarification would facilitate differentiation between rejection and non-rejection and identification of the mechanisms involved in the allograft rejection. Despite increasing evidence, further research is necessary to establish the clinical utility of extracellular vesicles in the diagnosis and monitoring of allograft rejection in kidney transplant recipients. Using extracellular vesicles as non-invasive biomarkers for diagnosis of kidney allograft rejection could have tremendous benefits in improving patient outcomes and reduce the need for invasive procedures.

Role of Extracellular Vesicles (EVs) in Mediating Antioxidant Response to a Glucoraphanin Rich Meal

Aging is associated with increasing oxidative stress and declining antioxidant defences, making the body susceptible to various chronic conditions. Cruciferous vegetables contain glucoraphanin, which is metabolised into sulforaphane (SFN) in the gut(1). SFN activates a mechanism enhancing antioxidant defences that may contribute to a reduction in risk of age-related chronic diseases. Our hypothesis proposes that consuming cruciferous vegetables releases hepatic extracellular vesicles (EVs), which carry with a cargo of antioxidant proteins, into the systemic circulation(2) that are transported to ‘at risk’ tissues. We seek to characterise the antioxidant protein content of EVs from blood samples of healthy human volunteers from a prospective dietary intervention study trial called (GLOBE). The GLOBE study employs a randomised, single-blind, two-arm crossover design and involves 12 healthy male or female adults aged &gt;55 years, at the time of enrolment, have a BMI in the range of 18.5-30 kg/m2, and have self-reported healthy (are not suffering from current illness like cancer, and gastrointestinal diseases including coeliac, Crohn’s, colitis, and irritable bowel syndrome) and not using any medications like antacids, laxatives and antibiotics which can interfere with normal digestive or metabolic processes. The dietary intervention consists of two commercially available treatments, one of which is a glucoraphanin-rich vegetable soup, while the other vegetable soup lacks glucoraphanin. Our initial focus lies in optimizing and standardizing a method for routinely characterizing EVs derived from healthy volunteers participating in dietary intervention studies. We specifically aim to extract EVs from a minimal plasma volume (2ml) using size exclusion chromatography (SEC). Subsequently, we intend to employ this method to analyse EVs obtained from 161 plasma samples collected from 12 participants during the GLOBE study for different time points like 0, 120, 240, 360, 480 min, 24 and 48 hr. Our goal is to gain insight into the role of EVs as part of the mechanisms by which consuming a moderate quantity of cruciferous vegetables may confer health benefits. Our research carries the potential to establish a standardised approach to the characterisation of EVs from healthy individuals which has several applications in nutrition research.

Changes Induced by P2X7 Receptor Stimulation of Human Glioblastoma Stem Cells in the Proteome of Extracellular Vesicles Isolated from Their Secretome.

Extracellular vesicles (EVs) are secreted from many tumors, including glioblastoma multiforme (GBM), the most common and lethal brain tumor in adults, which shows high resistance to current therapies and poor patient prognosis. Given the high relevance of the information provided by cancer cell secretome, we performed a proteomic analysis of microvesicles (MVs) and exosomes (EXOs) released from GBM-derived stem cells (GSCs). The latter, obtained from the brain of GBM patients, expressed P2X7 receptors (P2X7Rs), which positively correlate with GBM growth and invasiveness. P2X7R stimulation of GSCs caused significant changes in the EV content, mostly ex novo inducing or upregulating the expression of proteins related to cytoskeleton reorganization, cell motility/spreading, energy supply, protection against oxidative stress, chromatin remodeling, and transcriptional regulation. Most of the induced/upregulated proteins have already been identified as GBM diagnostic/prognostic factors, while others have only been reported in peripheral tumors. Our findings indicate that P2X7R stimulation enhances the transport and, therefore, possible intercellular exchange of GBM aggressiveness-increasing proteins by GSC-derived EVs. Thus, P2X7Rs could be considered a new druggable target of human GBM, although these data need to be confirmed in larger experimental sets.

Effects of Eribulin on the RNA Content of Extracellular Vesicles Released by Metastatic Breast Cancer Cells.

Extracellular vesicles (EVs) are small lipid particles secreted by almost all human cells into the extracellular space. They perform the essential function of cell-to-cell communication, and their role in promoting breast cancer progression has been well demonstrated. It is known that EVs released by triple-negative and highly aggressive MDA-MB-231 breast cancer cells treated with paclitaxel, a microtubule-targeting agent (MTA), promoted chemoresistance in EV-recipient cells. Here, we studied the RNA content of EVs produced by the same MDA-MB-231 breast cancer cells treated with another MTA, eribulin mesylate. In particular, we analyzed the expression of different RNA species, including mRNAs, lncRNAs, miRNAs, snoRNAs, piRNAs and tRNA fragments by RNA-seq. Then, we performed differential expression analysis, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, and miRNA-target identification. Our findings demonstrate the possible involvement of EVs from eribulin-treated cells in the spread of chemoresistance, prompting the design of strategies that selectively target tumor EVs.

Abstract B041: Modeling the malignant transformation of fallopian tube epithelium driven by extracellular vesicles cargos in an organ-on-chip microphysiological system

Abstract Epithelial ovarian cancer (EOC) remains the leading cause of death from gynecological malignancy and the fifth leading cause of all cancer-related deaths among American women. The most common and lethal subtype, high-grade serous ovarian cancer (HGSOC) tends to originate from the fallopian tube. Precursor lesions of HGSOC such as secretory cell expansion (SCE), the loss of PAX2, PTEN, p53 mutation and serous tubal intraepithelial carcinoma (STIC) are often found at the fallopian tube epithelium (FTE). However, the mechanism of how neoplastic lesions disseminate in FTE and eventually colonize the ovary is unclear. We are interested in understanding if ovarian cancer initiation in the FTE is mediated by extracellular vesicles (EVs), which are nanosized biovesicles secreted by all living cells. EVs serve as vehicles for nucleic acids, proteins, and lipids. Cancer cells commonly secrete more EVs than healthy cells and shift their EV cargos to promote tumorigenesis. The alteration of molecular content of tumor-derived EV can potentially indicate disease states and serve as a new class of cancer biomarkers. Hence, our hypothesis is that EV cargos secreted by tumor cells contain factors promoting preneoplastic lesions in the FTE. Our goal is to interrogate the role of EVs in the initiation of ovarian cancer in the FTE at the early stage, for which there is no adequate in vitro model. We created a microfluidic model using a 3D dynamic culture system termed PREDICT-MOS to enable the direct interaction of hFTE with EVs and the long-term culture of the fallopian tube explant. We are interested to 1) test the role of EVs to drive preneoplastic transformation of hFTE progenitor cells and 2) to test whether tumor derived-EVs shift EV proteins cargos secreted by hFTE. The hFTE cultured on PREDICT-MOS is directly exposed to OVCAR3-derived EVs and embedded for transcriptomic profiling utilizing GeoMx Digital Spatial Profiler to identify differentially expressed genes in both ciliated and secretory cells within the epithelium. In parallel, we are characterizing the hFTE EV protein content with or without OVCAR3 EV treatment to detect the alteration in hFTE cargos using mass spectrometry proteomics. Our preliminary transcriptomic data from 8 patient fallopian tube samples demonstrates the deregulation of pathways associated with inflammation (TNF and NF-kappa B signaling), immune suppression (PD-1 checkpoint signaling) and epithelium morphogenesis. In addition, we have identified 295 proteins as the results of the first proteomic characterization of hFTE-derived EV proteins, which can serve as a “baseline” to evaluate whether the fallopian tube shifts its sEV cargo during ovarian cancer carcinogenesis. Further validation of transcriptomic findings on translational level using immunohistochemistry and identification of OVCAR3 EV protein cargos that induce the transcriptomic changes are needed to establish the role of EVs in the initiation of HGSOC in the fallopian tube. This project is supported by the Ovarian Cancer Research Fund Alliance Program Grant. Citation Format: Didi Zha, Jared Sipes, Sagar Rayamajhi, Leonidas E. Bantis, Angela Russo, Harsh Pathak, Andrew K. Godwin, Joanna E. Burdette. Modeling the malignant transformation of fallopian tube epithelium driven by extracellular vesicles cargos in an organ-on-chip microphysiological system [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B041.

Exosomal small RNA profiling in first-trimester maternal blood explores early molecular pathways of preterm preeclampsia.

Preeclampsia (PE) is a severe obstetrical syndrome characterized by new-onset hypertension and proteinuria and it is often associated with fetal intrauterine growth restriction (IUGR). PE leads to long-term health complications, so early diagnosis would be crucial for timely prevention. There are multiple etiologies and subtypes of PE, and this heterogeneity has hindered accurate identification in the presymptomatic phase. Recent investigations have pointed to the potential role of small regulatory RNAs in PE, and these species, which travel in extracellular vesicles (EVs) in the circulation, have raised the possibility of non-invasive diagnostics. The aim of this study was to investigate the behavior of exosomal regulatory small RNAs in the most severe subtype of PE with IUGR. We isolated exosomal EVs from first-trimester peripheral blood plasma samples of women who later developed preterm PE with IUGR (n=6) and gestational age-matched healthy controls (n=14). The small RNA content of EVs and their differential expression were determined by next-generation sequencing and further validated by quantitative real-time PCR. We also applied the rigorous exceRpt bioinformatics pipeline for small RNA identification, followed by target verification and Gene Ontology analysis. Overall, >2700 small RNAs were identified in all samples and, of interest, the majority belonged to the RNA interference (RNAi) pathways. Among the RNAi species, 16 differentially expressed microRNAs were up-regulated in PE, whereas up-regulated and down-regulated members were equally found among the six identified Piwi-associated RNAs. Gene ontology analysis of the predicted small RNA targets showed enrichment of genes in pathways related to immune processes involved in decidualization, placentation and embryonic development, indicating that dysregulation of the induced small RNAs is connected to the impairment of immune pathways in preeclampsia development. Finally, the subsequent validation experiments revealed that the hsa_piR_016658 piRNA is a promising biomarker candidate for preterm PE associated with IUGR. Our rigorously designed study in a homogeneous group of patients unraveled small RNAs in circulating maternal exosomes that act on physiological pathways dysregulated in preterm PE with IUGR. Therefore, our small RNA hits are not only suitable biomarker candidates, but the revealed biological pathways may further inform us about the complex pathology of this severe PE subtype.

Extracellular vesicles produced by irradiated endothelial or Glioblastoma stem cells promote tumor growth and vascularization modulating tumor microenvironment

BackgroundGlioblastoma (GBM) is the most lethal primary brain tumor in adult, characterized by highly aggressive and infiltrative growth. The current therapeutic management of GBM includes surgical resection followed by ionizing radiations and chemotherapy. Complex and dynamic interplay between tumor cells and tumor microenvironment drives the progression and contributes to therapeutic resistance. Extracellular vesicles (EVs) play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.MethodsIn this study, we isolated by ultracentrifugation EVs from GBM stem-like cell (GSC) lines and human microvascular endothelial cells (HMVECs) exposed or not to ionizing irradiation. After counting and characterization, we evaluated the effects of exposure of GSCs to EVs isolated from endothelial cells and vice versa. The RNA content of EVs isolated from GSC lines and HMVECs exposed or not to ionizing irradiation, was analyzed by RNA-Seq. Periostin (POSTN) and Filamin-B (FLNB) emerged in gene set enrichment analysis as the most interesting transcripts enriched after irradiation in endothelial cell-derived EVs and GSC-derived EVs, respectively. POSTN and FLNB expression was modulated and the effects were analyzed by in vitro assays.ResultsWe confirmed that ionizing radiations increased EV secretion by GSCs and normal endothelial cells, affected the contents of and response to cellular secreted EVs. Particularly, GSC-derived EVs decreased radiation-induced senescence and promoted migration in HMVECs whereas, endothelial cell-derived EVs promoted tumorigenic properties and endothelial differentiation of GSCs. RNA-Seq analysis of EV content, identified FLNB and POSTN as transcripts enriched in EVs isolated after irradiation from GSCs and HMVECs, respectively. Assays performed on POSTN overexpressing GSCs confirmed the ability of POSTN to mimic the effects of endothelial cell-derived EVs on GSC migration and clonogenic abilities and transdifferentiation potential. Functional assays performed on HMVECs after silencing of FLNB supported its role as mediator of the effects of GSC-derived EVs on senescence and migration.ConclusionIn this study, we identified POSTN and FLNB as potential mediators of the effects of EVs on GSC and HMVEC behavior confirming that EVs play a crucial role in the intercellular communication by delivering bioactive molecules in the surrounding milieu modulating tumor microenvironment.

An Electrochemiluminescent Sensor Based on Glycosyl Imprinting and Aptamer for the Detection of Cancer-Related Extracellular Vesicles.

Cancer-related extracellular vesicles (EVs) are considered important biomarkers for cancer diagnosis because they can convey a large amount of information about tumor cells. In order to detect cancer-related EVs efficiently, an electrochemiluminescence (ECL) sensor for the specific identification and highly sensitive detection of EVs in the plasma of cancer patients was constructed based on dual recognitions by glycosyl-imprinted polymer (GIP) and aptamer. The characteristic glycosyl Neu5Ac-α-(2,6)-Gal-β-(1-4)-GlcNAc trisaccharide on the surface of EVs was used as a template molecule and 3-aminophenylboronic acid as a functional monomer to form a glycosyl-imprinted polymer by electropolymerization. After glycosyl elution, the imprinted film specifically recognized and adsorbed the EVs in the sample, and then the CD63 aptamer-bipyridine ruthenium (Aptamer-Ru(bpy)) was added to combine with the CD63 glycoprotein on the extracellular vesicle's surface, thus providing secondary recognition of the EVs. Finally, the EVs were quantitatively detected according to the ECL signal produced by the labeled bipyridine ruthenium. When more EVs were captured by the imprinted film, more probes were obtained after incubation, and the ECL signal was stronger. Under the optimized conditions, the ECL signal showed a good linear relationship with the concentration of EVs in the range of 9.5 × 102 to 9.5 × 107 particles/mL, and the limit of detection was 641 particles/mL. The GIP sensor can discriminate between the EV contents of cancer patients and healthy controls with high accuracy. Because of its affordability, high sensitivity, and ease of use, it is anticipated to be employed for cancer early detection and diagnosis.

Janus kinase inhibitors modify the fatty acid profile of extracellular vesicles and modulate the immune response

BackgroundJanus kinase inhibitors (jakinibs) are immunomodulators used for treating malignancies, autoimmune diseases, and immunodeficiencies. However, they induce adverse effects such as thrombosis, lymphocytosis, and neutropenia that could be mediated by extracellular vesicles (EVs). These particles are cell membrane-derived structures that transport cellular and environmental molecules and participate in intercellular communication. Jakinibs can modify the content of EVs and enable them to modulate the activity of different components of the immune response. Objectiveto evaluate the interactions between immune system components of healthy individuals and EVs derived from monocytic and lymphoid lineage cells generated in the presence of baricitinib (BARI) and itacitinib (ITA) and their possible effects. MethodsEVs were isolated from monocytes (M) and lymphocytes (L) of healthy individuals, as well as from U937 (U) and Jurkat (J) cells exposed to non-cytotoxic concentrations of BARI, ITA, and dimethyl sulfoxide (DMSO; vehicle control). The binding to and engulfment of EVs by peripheral blood leukocytes of healthy individuals were analyzed by flow cytometry using CFSE-stained EVs and anti-CD45-PeCy7 mAb-labeled whole blood. The effect of EVs on respiratory burst, T-cell activation and proliferation, cytokine synthesis, and platelet aggregation was evaluated. Respiratory burst was assessed in PMA-stimulated neutrophils by the dihydrorhodamine (DHR) test and flow cytometry. T-cell activation and proliferation and cytokine production were assessed in CFSE-stained PBMC cultures stimulated with PHA; expression of the T-cell activation markers CD25 and CD69 and T-cell proliferation were analyzed by flow cytometry, and the cytokine levels were quantified in culture supernatants by Luminex assays. Platelet aggregation was analyzed in platelet-rich plasma (PRP) samples by light transmission aggregometry. The EVs’ fatty acid (FA) profile was analyzed using methyl ester derivatization followed by gas chromatography. ResultsITA exposure during the generation of EVs modified the size of the EVs released; however, treatment with DMSO and BARI did not alter the size of EVs generated from U937 and Jurkat cells. Circulating neutrophils, lymphocytes, and monocytes showed a 2-fold greater tendency to internalize ITA-U-EVs than their respective DMSO control. The neutrophil respiratory burst was attenuated in greater extent by M-EVs than by L-EVs. Autologous ITA-M-EVs reduced T-cell proliferation by decreasing IL-2 levels and CD25 expression independently of CD69. A higher accumulation of pro-inflammatory cytokines was observed in PHA-stimulated PBMC cultures exposed to M-EVs than to L-EVs; this difference may be related to the higher myristate content of M-EVs. Platelet aggregation increased in the presence of ITA-L/M-EVs by a mechanism presumably dependent on the high arachidonic acid content of the vesicles. ConclusionsCellular origin and jakinib exposure modify the FA profile of EVs, enabling them, in turn, to modulate neutrophil respiratory burst, T-cell proliferation, and platelet aggregation. The increased T-cell proliferation induced by BARI-L/M-EVs could explain the lymphocytosis observed in patients treated with BARI. The higher proportion of arachidonic acid in the FA content of ITA-L/M-EVs could be related to the thrombosis described in patients treated with ITA. EVs also induced a decrease in the respiratory burst of neutrophils.

Therapeutic Applications of Extracellular Vesicles in Inflammatory Bowel Disease.

The treatment landscape for inflammatory bowel disease (IBD) has undergone substantial advancements with the introduction of biologics. However, a considerable number of patients either show an immediate lack of response or lose responsiveness over time, necessitating the development of innovative and effective treatment approaches. Extracellular vesicles (EVs) are small lipid bilayer-enclosed structures that facilitate cell-to-cell molecular transfer and are integral to the pathogenesis of IBD. They play pivotal roles in maintaining the integrity of the intestinal epithelial barrier and the expulsion of cellular metabolites. The potential use of EVs as drug carriers or therapeutic agents has opened up a plethora of clinical applications. This review investigates the creation and content of EVs, their role in IBD development, and advances in their isolation and analytical techniques. Furthermore, the therapeutic promise they hold for IBD is explored, along with the latest research on their roles as IBD drug delivery systems.

PRODUCTION OF CLINICAL-GRADE EXTRACELLULAR VESICLES (EVS) SECRETED BY INDUCED PLURIPOTENT STEM CELL-DERIVED MESENCHYMAL STEM CELLS AND MESENCHYMAL STEM CELLS FOR THE TREATMENT OF OSTEOARTHRITIS

Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and flow cytometry were used to identify EVs. We evaluated the biological effects of MSC and iMSC-derived EVs on human chondrocytes treated with IL-1α, to mimic the OA environment.In both cell types, from early to late passages, the amount of EVs detected by NTA increased significantly, EVs collected during cells expansion, retained tetraspanins (CD9, CD63 and CD81) expression. The anti-inflammatory activity of MSC-EVs was evaluated in vitro using OA chondrocytes, the expression of IL-6, IL-8 and COX-2 was significantly reduced after the treatment with hMSC-derived EVs isolated at early passage. The miRNA content of EVs was also investigated, we identify miRNA that are involved in specific biological function.At the same time, we defined the best culture conditions to maintain iMSC and define the best time window in which to isolate EVs with highest biological activity.In conclusion, a clinical grade serum-free medium was found to be suitable for the isolation and expansion of MSCs and iMSC with increased EVs production for therapeutic applications.Acknowledgments: This project has received funding from the European Union's Horizon 2020 research and innovation programme under grant agreement No 874671

The extracellular vesicle proteomes of Sorghum bicolor and Arabidopsis thaliana are partially conserved.

Plant extracellular vesicles (EVs) are membrane-bound organelles involved mainly in intercellular communications and defense responses against pathogens. Recent studies have demonstrated the presence of proteins, nucleic acids including small RNAs, and lipids along with other metabolites in plant EVs. Here, we describe the isolation and characterization of EVs from sorghum (Sorghum bicolor). Nanoparticle tracking analysis, dynamic light scattering, and cryo-electron tomography showed the presence of a heterogeneous population of EVs isolated from the apoplastic wash of sorghum leaves. Cryo-electron microscopy revealed that EVs had a median size of 110 nm and distinct populations of vesicles with single or multiple lipid bilayers and low or high amounts of contents. The heterogeneity was further supported by data showing that only a subset of EVs that were stained with a membrane dye, Potomac Gold, were also stained with the membrane-permeant esterase-dependent dye, calcein acetoxymethyl ester. Proteomic analysis identified 437 proteins that were enriched in multiple EV isolations, with the majority of these also found in the EV proteome of Arabidopsis (Arabidopsis thaliana). These data suggest a partial conservation of EV contents and function between the monocot, sorghum, and a distantly related eudicot, Arabidopsis.

Extracellular Vesicle Content Changes Induced by Melatonin Promote Functional Recovery of Pancreatic Beta Cells in Acute Pancreatitis.

Acute pancreatitis is an inflammatory disorder of the pancreas, which causes abnormal activation of immune cells. The macrophages were accumulated in pancreas and infiltrated into islets during the AP process to induce abnormal glucose metabolism. However, the role of macrophages in abnormal glucose metabolism remains understood. Extracellular vesicles act in the regulation of intercellular function, but whether EVs secreted by macrophages contribute to β cell failure and apoptosis in AP is unclear. Based on this, the aim of this study was to reveal the role of macrophages-EVs in AP and develop a treatment for symptoms of hyperglycemia in AP. The AP model was established and treated by various doses of melatonin to analyze the therapeutic effect. The accumulation and polarization of macrophages in the AP pancreas were observed, and the β cells were incubated with pancreatic derived EVs to analyze the role in β cell failure and apoptosis. The results showed that macrophages were recruited and polarized to M1 phenotype macrophages in the pancreas of AP mice, which obtained inflammatory EVs that contained specific miRNAs to induce β cell failure and apoptosis. Then, the EVs derived from M1 macrophages triggered β cell failure and apoptosis. Melatonin prevented polarization of macrophages to the M1 phenotype in vivo, which reduced the secretion of inflammatory EVs, changed the abundance of miRNAs in EVs, and therefore decreased inflammatory EV-mediated β cell failure and apoptosis. Our results demonstrate that similar to 20S proteasome inhibitor MG132, analyses indicated that melatonin prevented degradation of IκBα through the ubiquitylation pathway to restrict p50 subunits to the cytoplasm of macrophages, inhibited activation of the NF-κB pathway to downregulate the transcription of specific miRNAs, and reduced miRNA transport into EVs.

Optimization of plasma extracellular vesicle enrichment for in‐depth lipidomic profiling in Alzheimer’s disease

AbstractBackgroundLipid dyshomeostasis is associated with various disorders including Alzheimer’s disease (AD). The work from our laboratory (Su et al, JEV 2021), and others, suggests that extracellular vesicles (EVs) contain lipid biomarkers that could aid in the diagnosis of AD and diseases associated with impaired lipid metabolism.Knowing the lipid content of EVs is a key first step to discovering EV‐based lipid biomarkers in blood. Some studies have reported the lipid content of ‘’EVs’’ in blood. However, used EV isolation techniques that are known to co‐isolate free lipid and lipoproteins.Here we: 1) demonstrate the importance of size exclusion resin size to isolating highly enriched EVs for lipidomic analysis, 2) have developed a reference EV plasma lipidome for the research community and 3) determining if plasma EVs report on lipid dyshomeostasis in Alzheimer’s disease.MethodHuman plasma EVs were isolated using an assortment of commercially available kits or by density fractionation and size exclusion chromatography and characterised by western blot, transmission electron microscopy, and quantitative mass spectrometry based proteomic and lipidome analysis.ResultOf the high‐throughput EV isolation kits tested, the majority were unable to separate lipoproteins from EVs for the purposes of EV lipidomics. We then relied on comparing labor intensive methods (density gradient and size exclusion) to identify a combination of techniques that isolate the analytes of interest, EV proteins and lipids. Quantitative proteome and lipidome analysis strategies are being applied to highly enriched EVs with the goal of providing the reference lipidome of EVs in plasma and in Alzheimer’s disease.ConclusionTo enhance the confidence of EV lipid identification and reveal the lipidome of EVs in plasma, our study compared and identified techniques to enrich EVs and deplete non‐EV associated lipid and protein from human plasma. These findings serve as a reference resource and the foundation for research investigating plasma EV lipids as Alzheimer’s disease biomarkers.

Abstract LB_B21: Extracellular RNA signatures of mutant KRAS-driven lung cancer

Abstract RAS genes are the most frequently mutated oncogenes in cancer, yet the effects of oncogenic RAS signaling on the noncoding transcriptome remain unclear. We analyzed the transcriptomes of human airway and bronchial epithelial cells transformed with mutant KRAS to define the landscape of KRAS-regulated noncoding RNAs. We find that oncogenic KRAS signaling upregulates noncoding transcripts throughout the genome, many of which arise from transposable elements (TEs). These TE RNAs exhibit differential expression and are regulated by KRAB zinc-finger (KZNF) genes, which are broadly downregulated in mutant KRAS cells and lung adenocarcinomas in vivo. Using both affinity- and nanofiltration-based EV isolation approaches, we show that specific TE RNAs, lncRNAs, and mRNAs, some of which are prognostic for lung adenocarcinoma (LUAD) patient survival, are preferentially released in extracellular vesicles (EVs). Moreover, we find that inhibition of oncogenic KRAS signaling broadly reprograms the noncoding transcriptome, as evidenced by a substantial increase in TE RNA secretion. Mutant KRAS inhibition also increases the abundance of secreted lncRNAs and retained intron-containing transcripts, while decreasing the mRNA content of EVs. Notably, oncogenic KRAS signaling is required for the secretion of mRNAs from a set of 20 genes that are significantly associated with unfavorable clinical outcomes in LUAD. Our results indicate that mutant KRAS remodels the repetitive noncoding transcriptome, demonstrating the broad scope of intracellular and extracellular RNAs regulated by this oncogenic signaling pathway. Our study also suggests that both coding and noncoding RNAs that are secreted in EVs may serve as mutant KRAS-specific signatures for diagnosing lung cancer via minimally invasive RNA liquid biopsy technology, which we are currently developing as a companion diagnostic for mutant KRAS-targeting therapies. Citation Format: Sree Lakshmi Velandi Maroli, Roman E Reggiardo, Reem Khojah, Daniel H Kim. Extracellular RNA signatures of mutant KRAS-driven lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr LB_B21.

Biological Role of Extracellular Vesicles in Myeloid Neoplasms: A Systematic Review of the Current Literature

Background and aim: Extracellular vesicles (EVs) are lipid-bilayer membrane vesicles secreted by a variety of cells that carry bioactive molecules like proteins, lipids and nucleic acids. They are of increasing interest as they play an important biological role in cancer by a multitude of cell-extrinsic and cell-intrinsic mechanisms. However, the investigations of their biology in myeloid malignancies has not been systematically reviewed so far. Therefore, the aim of this review was to summarize the currently published literature in the field and provide an overview of their potential clinical applications. Method: We performed a systematic literature research with defined search strings in the Embase, Medline and PubMed Central databases and used a standardized data-extraction form to collect all relevant information. Results: 63 of 1529 studies met the inclusion/exclusion criteria and could be assigned to seven main investigational categories ( A). They comprised studies on their effect on i) prothrombotic activity (n=13), ii) tumor-microenvironment (n=14), iii) malignant transformation (n=5), iv) tumor progression (n=4), v) chemotherapy-resistance (n=5) as well as their use as vi) biomarkers (n=14) and vii) pharmacotherapeutic carriers (n=3) ( B). The most relevant biological mechanism is the transfer of microRNA (miRNA) by EVs from different clonal and non-clonal cells, which target the microenvironment, vascular system, as well as immune cells. All mechanisms identified in our review were assigned to one of the seven main categories and the most relevant are summarized in C: i) Prothrombotic activity: Leukaemic cells shed prothrombotic EVs containing tissue-factor (TF) and anionic phosphatidylserine (PS), which facilitate the recruitment of coagulation factors and a pro-coagulant state ( Zannoni, 2019). Reduction of EVs from platelets and leucocytes after chemotherapy attenuates this pro-coagulant state ( Van Aalderen, 2011). ii) Tumor microenvironment: EVs derived from MDS cells can disrupt the osteolineage differentiation of bone marrow mesenchymal stromal cells (BM-MSCs) and impair hematopoiesis ( Hayashi, 2022). iii) Malignant transformation: EVs containing BCR-ABL1 mRNA are integrated into BM-MSCs, which induce BCR-ABL1 expression and secretion of TGF-β1 leading to enhanced proliferation of BM-MSCs ( Fu, 2017). iv) Tumor progression: EVs derived from leukemic cells promote tumor progression by reducing apoptosis and tumor immune-surveillance. v) Chemotherapy resistance: AML-cells expel PEGylated liposomal doxorubicin (PLD) through EVs thus increasing their resistance ( Hekmatirad, 2021). vi) Disease biomarkers: AML patients at diagnosis have a higher number of EVs carrying the myeloblast markers CD34, CD117, CD33 and HLA-DR than AML patients in remission or healthy controls ( Tzoran, 2015). Based on differential abundancy and the content of proteins, lipids and nucleid acids EVs may be suitable as biomarkers for diagnosis, prognosis, and disease monitoring. vii) Pharmacotherapeutic role: Cytotoxic T-lymphocytes are more effective in killing the parental leukaemic cells after being exposed to their cell-derived EVs. The mechanism is related to leukemia antigen presentation on EVs to dendritic cells (DCs) using ICAM1 and Hsp70 ( Shen C, 2011). Finally, EVs can be used as carriers for bioactive compounds and may expand their potential use for cell-type specific delivery of drugs. Conclusions: EVs open the window for the future investigation of novel pathophysiological mechanisms that will improve our understanding of their role in the biology of myeloid malignancies, Moreover, they hold promise as potential biomarkers and drug carriers for cell-type specific treatment

The Proteomic Signature of Extracellular Vesicles from Sickle Cell Anemia Patients Provides Insights into Their Possible Role in the Pathophysiology of the Disease

Sickle cell anemia (SCA) is marked by hemolysis and vaso-occlusion, which contribute to various other complications observed in the disease. Extracellular vesicles (EVs), released by activated cells, serve as carriers of various markers, proteins, and RNA derived from their parent cells. These structures have the ability to transfer their cargo to the recipient cells, modulating their activity. Despite EVs involvement in physiological processes, there is growing evidence of their role in certain blood diseases, making them a subject of extensive research in recent years. In sickle cell disease (SCD), EVs have been previously associated with hemolysis markers, leg ulcers, and endothelial cell activation and several other authors have emphasized the significant role of EVs in its pathophysiology. However, despite these advances, the precise protein content of these EVs, which may hold the key to unraveling their involvement in disease complications, remains largely unexplored. Here, we aimed to characterize the proteomic profile of EVs from patients with SCA. We conducted a comparative analysis, comparing the differentially abundant proteins in the SCA group to those in the control group. Additionally, we focused on analyzing the distinct protein profiles that were uniquely present in the SCA group studied. Nine healthy volunteers (HC) and nineteen patients with SCA were included in the study, with three receiving no treatment, twelve undergoing hydroxyurea (HU) therapy, and four on HU+Crizanlizumab (CRIZ) combination treatment. EVs were isolated from plasma using a combination of differential centrifugation and size exclusion chromatography techniques, and their quantification was performed using nano-tracking analysis. The protein profile of EVs was assessed through mass spectrometry after sample digestion and desalination in a developed protocol for EVs research. The obtained data were analyzed using MaxQuant and Perseus software. To determine differentially abundant proteins between the HC and SCA groups, statistical tests such as ANOVA followed by Benjamini-Hochberg correction were applied, considering a significance level of p&amp;lt;0.05. Additionally, gene ontology analysis was conducted to identify the relevant biological processes (FDR &amp;lt;0,05) associated with the differentially abundant proteins. Firstly, we identified 41 unique proteins in the SCA group. The relevant biological processes associated with these proteins are primarily related to erythroid differentiation, inflammation, cellular response to oxidative stress, and the upregulation of signaling pathways mediated by TNF and other cytokines. These findings align with the characteristic pathophysiology of SCA. The proteins found in higher concentrations in EVs of SCA patients compared to HC group (p&amp;lt;0,05) were mainly related to endocytosis and exocytosis, inflammation, immune response and coagulation process. Moreover, we observed higher levels of hemoglobin chains in SCA EVs. The delta chain was increased in the SCA group with no treatment in relation to the HC group and the SCA group with HU. Beta and alpha chains were also found in increased levels in the SCA group without HU compared to the HC group, SCA with HU and SCA with HU+CRIZ. Additionally, both transferrin (TF) and the transferrin receptor (TFRC-CD71) were increased in all three subgroups of SCA patients compared to the HC group. The identification of various types of hemoglobin in these EVs holds significant importance. This observation aligns with the potential role of these structures in promoting the generation of reactive oxygen species (ROS), activating endothelial cells, and reducing nitric oxide levels in individuals with SCA. Such findings may provide valuable insights into the underlying mechanisms contributing to the pathophysiology of SCA and its associated complications. Moreover, our data shows higher levels of Tenascin-C in the EVs of SCA patients (all subgroups) (p&amp;lt;0,05), a protein related to tissue damage and inflammation that has not yet been described in the pathophysiology of the disease. In summary, we access, for the first time, the protein content of EVs from patients with SCA. The data described here are novel and could help to elucidate the putative functions of EVs in the context of SCA providing valuable insights for future investigations and potential therapeutic strategies.