Extracellular Vesicles Counts Research Articles

Wildfire-relevant woodsmoke and extracellular vesicles (EVs): Alterations in EV proteomic signatures involved in extracellular matrix degradation and tissue injury in airway organotypic models

Wildfires adversely impact air quality and public health worldwide. Exposures to wildfire smoke are linked to adverse health outcomes, including cardiopulmonary diseases. Critical research gaps remain surrounding the underlying biological pathways leading to wildfire-induced health effects. The regulation of intercellular communication and downstream toxicity driven by extracellular vesicles (EVs) is an important, understudied biological mechanism. This study investigated EVs following a wildfire smoke-relevant in vitro exposure. We hypothesized that woodsmoke (WS) would alter the proteomic content of EVs secreted in organotypic in vitro airway models. Exposures were carried out using a tri-culture model of alveolar epithelial cells, fibroblasts, and endothelial cells and a simplified co-culture model of alveolar epithelial cells and fibroblasts to inform responses across different cell populations. Epithelial cells were exposed to WS condensate and EVs were isolated from basolateral conditioned medium following 24 h exposure. WS exposure did not influence EV particle characteristics, and it moderately increased EV count. Exposure caused the differential loading of 25 and 35 proteins within EVs collected from the tri- and co-culture model, respectively. EV proteins involved in extracellular matrix degradation and wound healing were consistently modulated across both models. However, distinct proteins involved in the wound healing pathway were altered between models, suggesting unique but concerted efforts across cell types to communicate in response to injury. These findings demonstrate that a wildfire-relevant exposure alters the EV proteome and suggest an impact on EV-mediated intercellular communication. Overall, results demonstrate the viability of organotypic approaches in evaluating EVs to investigate exposure-induced biomarkers and underlying mechanisms. Findings also highlight the impact of differences in the biological complexity of in vitro models used to evaluate the effects of inhaled toxicants.

Extracellular vesicle transfer of miR-1 to adipose tissue modifies lipolytic pathways following resistance exercise.

Extracellular vesicles (EVs) have emerged as important mediators of intertissue signaling and exercise adaptations. In this human study, we provide evidence that muscle-specific microRNA-1 (miR-1) was transferred to adipose tissue via EVs following an acute bout of resistance exercise. Using a multimodel machine learning automation tool, we discovered muscle primary miR-1 transcript and CD63+ EV count in circulation as top explanatory features for changes in adipose miR-1 levels in response to resistance exercise. RNA-Seq and in-silico prediction of miR-1 target genes identified caveolin 2 (CAV2) and tripartite motif containing 6 (TRIM6) as miR-1 target genes downregulated in the adipose tissue of a subset of participants with the highest increases in miR-1 levels following resistance exercise. Overexpression of miR-1 in differentiated human adipocyte-derived stem cells downregulated these miR-1 targets and enhanced catecholamine-induced lipolysis. These data identify a potential EV-mediated mechanism by which skeletal muscle communicates with adipose tissue and modulates lipolysis via miR-1.

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

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

Choice of blood collection methods influences extracellular vesicles counts and miRNA profiling.

Circulating RNAs have been investigated systematically for over 20years, both as constituents of circulating extracellular vesicles (EVs) or, more recently, non-EV RNA carriers, such as exomeres and supermeres. The high level of variability and low reproducibility rate of EV/extracellular RNA (exRNA) results generated even on the same biofluids promoted several efforts to limit pre-analytical variability by standardizing sample collection and sample preparation, along with instrument validation, setup and calibration. Anticoagulants (ACs) are often chosen based on the initial goal of the study and not necessarily for the later EV and/or exRNA analyses. We show the effects of blood collection on EV size, abundance, and antigenic composition, as well on the miRNAs. Our focus of this work was on the effect of ACs on the number and antigenic composition of circulating EVs and on a set of circulating miRNA species, which were shown to be relevant as disease markers in several cancers and Alzheimer's disease. Results show that while the number of plasma EVs, their relative size, and post-fluorescence labeling profile varied with each AC, their overall antigenic composition, with few exceptions, did not change significantly. However, the number of EVs expressing platelet and platelet-activation markers increased in serum samples. For overall miRNA expression levels, EDTA was a better AC, although this may have been associated with stimulation of cells in the blood collection tube. Citrate and serum rendered better results for a set of miRNAs that were described as circulating markers for Alzheimer's disease, colon, and papillary thyroid cancers.

Thrombin Generation Is Associated With Extracellular Vesicle and Leukocyte Lipid Membranes in Atherosclerotic Cardiovascular Disease.

Clotting, leading to thrombosis, requires interactions of coagulation factors with the membrane aminophospholipids (aPLs) phosphatidylserine and phosphatidylethanolamine. Atherosclerotic cardiovascular disease (ASCVD) is associated with elevated thrombotic risk, which is not fully preventable using current therapies. Currently, the contribution of aPL to thrombotic risk in ASCVD is not known. Here, the aPL composition of circulating membranes in ASCVD of varying severity will be characterized along with the contribution of external facing aPL to plasma thrombin generation in patient samples. Thrombin generation was measured using a purified factor assay on platelet, leukocyte, and extracellular vesicles (EVs) from patients with acute coronary syndrome (n=24), stable coronary artery disease (n=18), and positive risk factor (n=23) and compared with healthy controls (n=24). aPL composition of resting/activated platelet and leukocytes and EV membranes was determined using lipidomics. External facing aPLs were detected on EVs, platelets, and leukocytes, elevating significantly following cell activation. Thrombin generation was higher on the surface of EVs from patients with acute coronary syndrome than healthy controls, along with increased circulating EV counts. Thrombin generation correlated significantly with externalized EV phosphatidylserine, plasma EV counts, and total EV membrane surface area. In contrast, aPL levels and thrombin generation from leukocytes and platelets were not impacted by disease, although circulating leukocyte counts were higher in patients. The aPL membrane of EV supports an elevated level of thrombin generation in patient plasma in ASCVD. Leukocytes may also play a role although the platelet membrane did not seem to contribute. Targeting EV formation/clearance and developing strategies to prevent the aPL surface of EV interacting with coagulation factors represents a novel antithrombotic target in ASCVD.

Spinal cord injury disrupts plasma extracellular vesicles cargoes leading to neuroinflammation in the brain and neurological dysfunction in aged male mice

Aged individuals with spinal cord injury (SCI) are prevalent with increased mortality and worse outcomes. SCI can cause secondary brain neuroinflammation and neurodegeneration. However, the mechanisms contributing to SCI-induced brain dysfunction are poorly understood. Cell-to-cell signaling through extracellular vesicles (EVs) has emerged as a critical mediator of neuroinflammation, including at a distance through circulation. We have previously shown that SCI in young adult (YA) male mice leads to robust changes in plasma EV count and microRNAs (miRs) content. Here, our goal was to investigate the impact of old age on EVs and brain after SCI. At 24 h post-injury, there was no difference in particle count or size distribution between YA and aged mice. However, aged animals increased expression of EV marker CD63 with SCI. Using the Fireplex® miRs assay, Proteomics, and mass spectrometry-based Lipidomics, circulating EVs analysis identified distinct profiles of miRs, proteins, and lipid components in old and injury animals. In vitro, plasma EVs from aged SCI mice, at a lower concentration comparable to those of YA SCI mice, induced the secretion of pro-inflammatory cytokines and neuronal apoptosis. Systemic administration of plasma EVs from SCI animals was sufficient to impair general physical function and neurological function in intact animals, which is associated with pro-inflammatory changes in the brain. Furthermore, plasma EVs from young animals had rejuvenating effects on naïve aged mice. Collectively, these studies identify the critical changes in circulating EVs cargoes after SCI and in aged animals and support a potential EV-mediated mechanism for SCI-induced brain changes.

Increased secretion of adipocyte-derived extracellular vesicles is associated with adipose tissue inflammation and the mobilization of excess lipid in human obesity

BackgroundObesity is a worldwide epidemic characterized by adipose tissue (AT) inflammation. AT is also a source of extracellular vesicles (EVs) that have recently been implicated in disorders related to metabolic syndrome. However, our understanding of mechanistic aspect of obesity’s impact on EV secretion from human AT remains limited.MethodsWe investigated EVs from human Simpson Golabi Behmel Syndrome (SGBS) adipocytes, and from AT as well as plasma of subjects undergoing bariatric surgery. SGBS cells were treated with TNFα, palmitic acid, and eicosapentaenoic acid. Various analyses, including nanoparticle tracking analysis, electron microscopy, high-resolution confocal microscopy, and gas chromatography–mass spectrometry, were utilized to study EVs. Plasma EVs were analyzed with imaging flow cytometry.ResultsEVs from mature SGBS cells differed significantly in size and quantity compared to preadipocytes, disagreeing with previous findings in mouse adipocytes and indicating that adipogenesis promotes EV secretion in human adipocytes. Inflammatory stimuli also induced EV secretion, and altered EV fatty acid (FA) profiles more than those of cells, suggesting the role of EVs as rapid responders to metabolic shifts. Visceral AT (VAT) exhibited higher EV secretion compared to subcutaneous AT (SAT), with VAT EV counts positively correlating with plasma triacylglycerol (TAG) levels. Notably, the plasma EVs of subjects with obesity contained a higher number of adiponectin-positive EVs than those of lean subjects, further demonstrating higher AT EV secretion in obesity. Moreover, plasma EV counts of people with obesity positively correlated with body mass index and TNF expression in SAT, connecting increased EV secretion with AT expansion and inflammation. Finally, EVs from SGBS adipocytes and AT contained TAGs, and EV secretion increased despite signs of less active lipolytic pathways, indicating that AT EVs could be involved in the mobilization of excess lipids into circulation.ConclusionsWe are the first to provide detailed FA profiles of human AT EVs. We report that AT EV secretion increases in human obesity, implicating their role in TAG transport and association with adverse metabolic parameters, thereby emphasizing their role in metabolic disorders. These findings promote our understanding of the roles that EVs play in human AT biology and metabolic disorders.

Plasma extracellular vesicles proteomics in meningioma patients

Tumor derived Extracellular vesicles (EVs) in circulating system may contain tumor-specific markers, and EV detection in body fluids could become an important tool for early tumor diagnosis, prognosis assessment. Meningiomas are the most common benign intracranial tumors, few studies have revealed specific protein markers for meningiomas from patients’ body fluids. In this study, using proximity labeling technology and non-tumor patient plasma as a control, we detected protein levels of EVs in plasma samples from meningioma patients before and after surgery. Through bioinformatics analysis, we discovered that the levels of EV count and protein count in meningioma patients were significantly higher than those in healthy controls, and were significantly decreased postoperatively. Among EV proteins in meningioma patients, the levels of MUC1, SIGLEC11, E-Cadherin, KIT, and TASCTD2 were found not only significantly elevated than those in healthy controls, but also significantly decreased after tumor resection. Moreover, using publicly available GEO databases, we verified that the mRNA level of MUC1, SIGLEC11, and CDH1 in meningiomas were significantly higher in comparison with normal dura mater tissues. Additionally, by analyzing human meningioma specimens collected in this study, we validated the protein levels of MUC1 and SIGLEC11 were significantly increased in WHO grade 2 meningiomas and were positively correlated with tumor proliferation levels. This study indicates that meningiomas secret EV proteins into circulating system, which may serve as specific markers for diagnosis, malignancy predicting and tumor recurrent assessment.

#1984 Extracellular vesicles drive loss of endothelial homeostasis in pediatric chronic kidney disease

Abstract Background and Aims Cardiovascular disease (CVD) is the main cause of mortality in chronic kidney disease (CKD). However, the pathogenesis of CVD in CKD remains incompletely understood. Extracellular vesicles (EVs) emerged as mediators of inter-organ cross-talk and endothelial EVs (EC-EVs) were previously associated with CVD. We hypothesized that CKD drives endothelial EV release in CKD and that these EC-EVs drive CVD in CKD. Method A cohort of 94 children (mean age 10.9 years) at different stages of CKD (patients with or without dialysis and after kidney transplantation (KTx)) and age-matched healthy donors was recruited, offering the unique opportunity to analyze cardiovascular effects of CKD and EV dynamics in absence of age-related comorbidities. Thorough characterization of plasma EVs by electron microscopy, nanoparticle tracking analysis (NTA), flow cytometry and miRNA sequencing was carried out. Functional properties of patient EVs and CKD-specific EV release mechanisms were tested in vitro. Results EC-EVs were elevated in hemodialysis (HD) patients (as measured by flow cytometry) while total EV counts showed higher concentrations in peritoneal dialysis patients (according to NTA). Longitudinal follow-ups revealed that EC-EVs decreased after receiving KTx. EV small RNA sequencing showed lower abundance of 30 microRNAs in EVs from CKD patients, most pronounced in HD patients compared to both healthy donors and KTx recipients. Downregulation of let-7d-5p, miR-19a-3p, miR-24-3p, miR-103a-3p and miR-142-3p was confirmed using RT-qPCR. Gene enrichment analyses predicted angiogenesis and smooth muscle cell (SMC) proliferation as targets of differentially regulated miRNAs in CKD. In vitro, angiogenesis of Human Umbilical Vein Endothelial Cells (HUVECs) was decreased upon treatment with CKD EVs as indicated by decreased vascular tube formation, endothelial migration and proliferation while SMC proliferation remained unchanged. To assess the putative role of uremic toxins for EV release in CKD, we focused on tryptophan metabolites and performed targeted plasma metabolomics that showed stage-dependent increases of uremic toxins of indole and kynurenine pathways in children with CKD, with highest levels in dialysis patients and almost normal levels after KTx. Uremic toxins indoxyl sulfate, formylkynurenine and xanthurenic acid correlated positively with EC-EVs in our cohort, but were not sufficient to trigger EV release from aortic endothelial cells alone. However, high shear stress together with indoxyl sulfate increased EV release from venous endothelial cells corresponding to higher endothelial EV concentrations in HD patients with arterio-venous fistulas (AVF) and recapitulated EV miRNA changes in vitro observed in CKD in vivo (lower abundance of let-7d-5p and miR-24-3p). Conclusion Endothelial EV release and decreased EV miRNAs associate with advanced CKD while increased endothelial shear stress and indoxyl sulfate form potential causative factors of altered EV phenotype. CKD EVs inhibit angiogenesis in vitro, potentially caused by dysregulated EV miRNAs that could form novel therapeutical targets to reduce CVD burden in CKD.

Hyaluronic Acid and Large Extracellular Vesicles (EVs) in Synovial Fluid and Plasma of Patients With End-Stage Arthritis: Positive Association of EVs to Joint Pain.

Hyaluronic acid (HA) in synovial fluid (SF) contributes to boundary lubrication with altered levels in osteoarthritis (OA) and rheumatoid arthritis (RA). SF extracellular vesicles (EVs) may participate in arthritis by affecting inflammation and cartilage degradation. It remains unknown whether HA and EVs display joint-specific alterations in arthritic SFs. We investigated the numbers and characteristics of HA-particles and large EVs in SF from knees and shoulders of 8 OA and 8 RA patients and 8 trauma controls, and in plasma from 10 healthy controls and 11 knee OA patients. The plasma and SF HA concentrations were determined with a sandwich-type enzyme-linked sorbent assay, and EVs and HA-particles were characterized from plasma and unprocessed and centrifuged SFs with confocal microscopy. The data were compared according to diagnosis, location, and preanalytical processing. The main findings were: (1) OA and RA SFs can be distinguished from trauma joints based on the distinctive profiles of HA-particles and large EVs, (2) there are differences in the SF HA and EV characteristics between shoulder and knee joints that could reflect their dissimilar mobility, weight-bearing, and shock absorption properties, (3) EV counts in SF and plasma can positively associate with pain parameters independent of age and body adiposity, and (4) low-speed centrifugation causes alterations in the features of HA-particles and EVs, complicating their examination in the original state. Arthritis and anatomical location can affect the characteristics of HA-particles and large EVs that may have potential as biomarkers and effectors in joint degradation and pain.

Red blood cell transfusion-related dynamics of extracellular vesicles in intensive care patients: a prospective subanalysis

Extracellular vesicles (EVs) accumulate during packed red blood cell (PRBC) storage. To date, the involvement of EVs in transfusion-related immunomodulation (TRIM) has not been prospectively evaluated in intensive care unit (ICU) patients. This was a prospective subanalysis of a recent observational feasibility study in postoperative ICU patients after: (1) open aortic surgery (Aorta), (2) bilateral lung transplantation (LuTx), and (3) other types of surgery (Comparison). Patient plasma was collected three times each before and after leukoreduced PRBC transfusion at 30-min intervals. The total number of EVs and EVs derived from erythrocytes (EryEVs), total platelets (total PEVs), activated platelets, granulocytes (GEVs), monocytes, and myeloid cells in PRBC samples and patient plasma were analyzed by flow cytometry. Statistical analysis was performed by Spearman’s correlation test, linear mixed models and pairwise comparisons by Wilcoxon matched-pairs test. Twenty-three patients (Aorta n = 5, LuTx n = 9, Comparison n = 9) were included in the final analysis. All EV subgroups analyzed were detectable in all PRBCs samples (n = 23), but concentrations did not correlate with storage time. Moreover, all EVs analyzed were detectable in all plasma samples (n = 138), and EV counts were consistent before transfusion. Concentrations of total EVs, EryEVs, total PEVs, and GEVs increased after transfusion compared with baseline in the entire cohort but not in specific study groups. Furthermore, the change in plasma EV counts (total EVs and EryEVs) after transfusion correlated with PRBC storage time in the entire cohort. Extracellular vesicles were detectable in all PRBC and plasma samples. Individual EV subtypes increased after transfusion in the entire cohort, and in part correlated with storage duration. Future clinical studies to investigate the role of EVs in TRIM are warranted and should anticipate a larger sample size.Trial registration: Clinicaltrials.gov: NCT03782623.

Characteristics of chemically induced liver progenitors derived from a pig model of metabolic dysfunction-associated steatotic liver disease.

We previously reported the efficacy of chemically induced liver progenitors (CLiP) as a source of cells for transplantation in patients with liver disease. This study aimed to characterize CLiP derived from steatotic livers using a pig model for future clinical applications. Livers were removed from miniature pigs with diet-induced steatosis and normal livers by laparoscopic hepatectomy. Mature hepatocytes (MH) isolated from the livers of each group were cultured in differentiation medium composed of Y-27632, A-83-01, and CHIR99021 (YAC medium). The characteristics of CLiP, including liver-specific function, proliferative capacity in vivo, and extracellular vesicles (EVs) production, were evaluated. Although CLiP in both groups expressed hepatic progenitor cell markers (Epithelial cell adhesion molecule and Trophoblast cell surface antigen 2), the proliferative potential was higher for the disease group than the healthy group. In contrast, markers of functional MH after re-differentiation were only detected in the healthy group. Both groups showed high cell viability and the ability to differentiate into albumin-positive cells in vivo. EVs counts were lower in disease-derived CLiP than in the normal group; however, there were no differences in microRNA expression within EVs. Using a pig model, CLiP was successfully produced from a liver that reproduced steatotic liver disease. Although there were slightly fewer EVs from CLiP in the disease group than in the normal liver group, the in vivo proliferative capacity of CLiP was high. Therefore, CLiP induced in the steatotic liver are a promising source for cell therapy in patients with liver disease.

Exploring Coagulation and Fibrinolysis Activation As Mechanisms of Sepsis-Related Tissue Damage in Patients with Severe Neutropenia and Thrombocytopenia Secondary to Hematological Malignancies

Sepsis is a life-threatening end-organ dysfunction resulting from dysregulated host responses to infection with physiopathology not fully understood. Yet, it has been long recognized that platelets and neutrophil extracellular traps collaborate to promote secondary tissue damage. Interestingly, patients with hematological malignancies and febrile neutropenia (FN) secondary to chemotherapy, with neutrophils and platelet counts frequently around zero, develop sepsis and septic shock, raising questions about which mechanisms are involved. Here, we show that extracellular vesicles (EVs) and activation of the fibrinolytic system are two potential mediators of tissue damage in sepsis among patients with very low platelets and neutrophil counts. The study population consisted of patients with hematologic malignancies who underwent antineoplastic treatment and that presented neutrophil counts < 500/μl and temperature ≥ 37.5 °C. Sepsis was defined as an increase of at least 2 points in the Sequential Organ Failure Assessment (SOFA) score and septic shock was defined by the need for vasoactive drugs after adequate volume infusion. Samples were obtained after FN diagnosis, at sepsis onset, and 72 h thereafter. A control group of patients with hematological malignancies and chemotherapy-induced neutropenia without fever was also included. Forty-two subjects, both genders, aged from 21 to 71 years were included and categorized into 4 groups, referred to: FN (n=18), FN + sepsis (n=10), FN + sepsis 72h (n=9), and the control group (n=18). The clinical and laboratory characteristics of these groups are described in Table 1. We first evaluated whether the release of EVs could be associated with the development of sepsis in these patients. EVs were characterized and enumerated from platelet-poor plasma (PPP) obtained from all groups and then separated through high-speed centrifugation (30 min, 20,000 g, 4ºC). EVs were marked with CD146 and 144 for endothelial cells; CD66b for granulocytes; CD14 and CD68 for monocytes, CD41 for platelets, and CD142 for tissue factor (TF) expression. Lactadherin and calcein were used to identify EVs according to size and integrity. Samples were acquired in a flow cytometer, Cytoflex S, with 30 μL/min flow rate and calibration microbeads were used to identify EV of specific sizes (Rosetta). The main findings are shown in Table 2. The main results were an increase in monocyte-derived EVs in patients with FN after sepsis onset (FN + sepsis 72h), compared to all other groups (p=0.01). Moreover, the expression of TF + was higher in these EVs (FN + sepsis 72h) when compared to EVs obtained at sepsis onset (FN + sepsis): 0.50 (IQR 0.29 - 1.10) events/μL versus 0.28 (IQR 0.12 - 0.56) in FN + Sepsis (p=0.03). Additionally, an increase in endothelial (CD146 +) and granulocyte-derived EVs were observed at the same time-point compared to FN and the control group. Of note, monocyte-derived EVs counts were not correlated with monocyte cell counts (Rs= 0.03; p=0.94). Given that coagulation activation plays a role in the immunothrombotic cascade known to mediate tissue damage in sepsis, we also explored whether thrombin and plasmin generation were increased in these patients. Briefly, the assay used PPP and two independent fluorometric substrates for thrombin (Boc-Val-Pro-Arg-MCA) and plasmin (Boc-Glu-Lys-Lys-MCA) in a 96-well plate, lately initiated by the addition of TF (10 pM). The plate was read in a fluorometric spectrophotometer for 4 h. Results are shown in Table 2, and no differences were observed in thrombin generation. However, the area under the curve of plasmin generation, which could be interpreted as a proxy of the plasma fibrinolytic potential, was lower in both FN + sepsis and FN + sepsis 72 h when compared to FN and the control group. Our findings reveal that EVs might present a role in the pathogenesis of sepsis in a scenario where cells known to be critical to this process are virtually absent. The increase of TF + EVs from monocytes, which has been demonstrated in classical sepsis, seems to be relevant even in this context, warranting additional studies using functional assays to further explore this mechanism of tissue damage. Our results also demonstrate that hypofibrinolysis is a characteristic of FN-associated sepsis, suggesting that deregulation of the fibrinolytic system is at least in part independent of the participation of granulocytes and platelets in these patients.

Sexually dimorphic extracellular vesicle responses after chronic spinal cord injury are associated with neuroinflammation and neurodegeneration in the aged brain

BackgroundMedical advances have made it increasingly possible for spinal cord injury (SCI) survivors to survive decades after the insult. But how SCI affects aging changes and aging impacts the injury process have received limited attention. Extracellular vesicles (EVs) are recognized as critical mediators of neuroinflammation after CNS injury, including at a distance from the lesion site. We have previously shown that SCI in young male mice leads to robust changes in plasma EV count and microRNA (miR) content. Here, our goal was to investigate the impact of biological sex and aging on EVs and brain after SCI.MethodsYoung adult age-matched male and female C57BL/6 mice were subjected to SCI. At 19 months post-injury, total plasma EVs were isolated by ultracentrifugation and characterized by nanoparticle tracking analysis (NTA). EVs miR cargo was examined using the Fireplex® assay. The transcriptional changes in the brain were assessed by a NanoString nCounter Neuropathology panel and validated by Western blot (WB) and flow cytometry (FC). A battery of behavioral tests was performed for assessment of neurological function.ResultsTranscriptomic changes showed a high number of changes between sham and those with SCI. Sex-specific changes were found in transcription networks related to disease association, activated microglia, and vesicle trafficking. FC showed higher microglia and myeloid counts in the injured tissue of SCI/Female compared to their male counterparts, along with higher microglial production of ROS in both injured site and the brain. In the latter, increased levels of TNF and mitochondrial membrane potential were seen in microglia from SCI/Female. WB and NTA revealed that EV markers are elevated in the plasma of SCI/Male. Particle concentration in the cortex increased after injury, with SCI/Female showing higher counts than SCI/Male. EVs cargo analysis revealed changes in miR content related to injury and sex. Behavioral testing confirmed impairment of cognition and depression at chronic time points after SCI in both sexes, without significant differences between males and females.ConclusionsOur study is the first to show sexually dimorphic changes in brain after very long-term SCI and supports a potential sex-dependent EV-mediated mechanism that contributes to SCI-induced brain changes.

CRISPR/Cas13a-Based MicroRNA Detection in Tumor-Derived Extracellular Vesicles.

MicroRNAs (miRNAs) in extracellular vesicles (EVs) play essential roles in cancer initiation and progression. Quantitative measurements of EV miRNAs are critical for cancer diagnosis and longitudinal monitoring. Traditional PCR-based methods, however, require multi-step procedures and remain as bulk analysis. Here, the authors introduce an amplification-free and extraction-free EV miRNA detection method using a CRISPR/Cas13a sensing system. CRISPR/Cas13a sensing components are encapsulated in liposomes and delivered them into EVs through liposome-EV fusion. This allows for accurately quantify specific miRNA-positive EV counts using 1 × 108 EVs. The authors show that miR-21-5p-positive EV counts are in the range of 2%-10% in ovarian cancer EVs, which is significantly higher than the positive EV counts from the benign cells (<0.65%). The result show an excellent correlation between bulk analysis with the gold-standard method, RT-qPCR. The authors also demonstrate multiplexed protein-miRNA analysis in tumor-derived EVs by capturing EpCAM-positive EVs and quantifying miR-21-5p-positive ones in the subpopulation, which showsignificantly higher counts in the plasma of cancer patients than healthy controls. The developed EV miRNA sensing system provides the specific miRNA detection method in intact EVs without RNA extraction and opens up the possibility of multiplexed single EV analysis for protein and RNA markers.

Contribution of Hepatic Steatosis–Intensified Extracellular Vesicle Release to Aggravated Inflammatory Endothelial Injury in Liver-Specific Asah1 Gene Knockout Mice

To study the mechanism by which nonalcoholic fatty liver disease (NAFLD) contributes to vascular endothelial Nod-like receptor pyrin domain 3 (NLRP3) inflammasome activation and neointima hyperplasia, NAFLD was established in high-fat diet (HFD)-treated Asah1fl/fl/Albcre (liver-specific deletion of the acid ceramidase gene Asah1) mice. Compared with Asah1 flox [Asah1fl/fl/wild type (WT)] and wild-type (WT/WT) mice, Asah1fl/fl/Albcre mice exhibited significantly enhanced ceramide levels and lipid deposition on HFD in the liver. Moreover, Asah1fl/fl/Albcre mice showed enhanced expression of extracellular vesicle (EV) markers, CD63 and annexin II, but attenuated lysosome-multivesicular body fusion. All these changes were accompanied by significantly increased EV counts in the plasma. In a mouse model of neointima hyperplasia, liver-specific deletion of the Asah1 gene enhanced HFD-induced neointima proliferation, which was associated with increased endothelial NLRP3 inflammasome formation and activation and more severe endothelial damage. The EVs isolated from plasma of Asah1fl/fl/Albcre mice on HFD were found to markedly enhance NLRP3 inflammasome formation and activation in primary cultures of WT/WT endothelial cells compared with those isolated from WT/WT mice or normal diet-treated Asah1fl/fl/Albcre mice. These results suggest that the acid ceramidase/ceramide signaling pathway controls EV release from the liver, and its deficiency aggravates NAFLD and intensifies hepatic EV release into circulation, which promotes endothelial NLRP3 inflammasome activation and consequent neointima hyperplasia in the mouse carotid arteries.

Diagnosis of Extracellular Vesicles in Cardiovascular and Metabolic Diseases.

Early detection and identification of those with or at increased risk for cardiovascular disease (CVD) and metabolic dysfunction is crucial for improving disease management and prognosis. Given the complex, multifactorial nature of the pathogenesis of the CVD and metabolic dysfunction, it is essential to have biomarkers that encompass the multiple facets of disease development. The uniquely ubiquitous nature and functionality of extracellular vesicles (EVs) in various disease pathologies can provide novel insight into both diagnosis and prognosis while further improving assessments used in clinical and research practice. Herein we summarize the use of EV count and content (including miRNA and protein) in diagnosis of CVD, obesity, metabolic syndrome, and type 2 diabetes (T2D), as well as highlight the potential utility for enhancing determination of prognosis and long-term complications in these clinical populations. Although the results are promising, future work is needed in both methodology and in relation to other factors such as sex and medications, in order to apply these findings in clinical practice.

Circulating Extracellular Vesicles Are Increased in Newly Diagnosed Celiac Disease Patients.

Extracellular vesicles (EVs) are a class of circulating entities that are involved in intercellular crosstalk mechanisms, participating in homeostasis maintenance, and diseases. Celiac disease is a gluten-triggered immune-mediated disorder, characterized by the inflammatory insult of the enteric mucosa following local lymphocytic infiltration, resulting in villous atrophy. The goal of this research was the assessment and characterization of circulating EVs in celiac disease patients, as well as in patients already on an adequate gluten-free regimen (GFD). For this purpose, a novel and validated technique based on polychromatic flow cytometry that allowed the identification and enumeration of different EV sub-phenotypes was applied. The analysis evidenced that the total, annexin V+, leukocyte (CD45+), and platelet (CD41a+) EV counts were significantly higher in both newly diagnosed celiac disease patients and patients under GFD compared with the healthy controls. Endothelial-derived (CD31+) and epithelial-derived (EpCAM+) EV counts were significantly lower in subjects under gluten exclusion than in celiac disease patients, although EpCAM+ EVs maintained higher counts than healthy subjects. The numbers of EpCAM+ EVs were a statistically significant predictor of intraepithelial leukocytes (IEL). These data demonstrate that EVs could represent novel and potentially powerful disease-specific biomarkers in the context of celiac disease.

Circulating extracellular vesicles as a predictive biomarker for acute graft-versus-host disease

The diagnosis and management of graft-versus-host disease (GVHD) have remained important challenges in allogeneic stem cell transplantation (allo-SCT). Novel diagnostic methods and therapeutic interventions are needed to further improve on patient outcomes. Extracellular vesicles (EV) are microvesicles formed by the inversion of the phospholipid bilayer of different cellular subtypes and have been described as biomarkers of cellular damage, activation, and intercellular signaling in numerous clinical scenarios. We studied the association between the levels of EV and the incidence of acute GVHD (aGVHD). Forty patients undergoing allo-SCT for hematological malignancies had their plasma collected at neutrophil engraftment. Using flow cytometry combined with fluorescent beads, the total circulating EV count (TEV) was established with annexin V positivity; CD61 positivity was used for platelet-derived EV (PEV), and CD235 positivity, for erythrocyte-derived EV (EryEV). TEV counts greater than 516/μL were associated with a higher cumulative incidence (CI) of grade II to IV aGVHD (54% vs. 21%; p=0.02), as were EryEV counts above 357 /μL (CI of aGVHD: 59% vs. 26%; p=0.04). In patients who are exposed to reduced intensity conditioning (RIC), stronger associations of both high TEV and EryEV counts with aGVHD were observed (77% vs. 22%; p=0.003 and 89% vs. 27%; p=0.002, respectively). PEV levels were not associated with the risk of aGVHD. Our data suggest that the measurement of cell-derived EV at engraftment can be used as a preemptive biomarker for acute GVHD.

BIOM-52. DIAGNOSTIC POTENTIAL OF EXTRACELLULAR VESICLES IN MENINGIOMA PATIENTS

Abstract Standard monitoring after meningioma resection relies on serial MRI examinations, which are time-consuming, expensive and provide no information on molecular alterations that may indicate progression towards a more aggressive tumor. Extracellular vesicles (EVs) secreted by tumor cells play an important role in cell-cell communication, and tumor-derived EVs circulating in patient blood can serve as biomarkers. We investigated the potential role of plasma EVs in meningioma patients for tumor detection and determined whether EVs secreted by meningioma cells reflect epigenetic, genomic and proteomic alterations of original tumors. EV concentrations were quantified in patient plasma (n = 46). Short-term meningioma cultures were established (n = 26) and secreted EVs were isolated. Methylation and copy number profiling was performed using 850k arrays, and mutations were identified by targeted gene panel sequencing. Differential quantitative mass spectrometry was employed for proteomic analysis. We found that the levels of circulating EVs were significantly elevated in meningioma patients compared to healthy individuals, and that plasma EV concentrations correlated with malignancy grade and extent of peritumoral edema. Postoperatively, EV counts dropped to normal levels, and the magnitude of the postoperative decrease was associated with extent of tumor resection (Simpson grade). Methylation profiling of EV-DNA allowed correct tumor classification as meningioma in all investigated cases, and accurate methylation subclass assignment in nearly all cases. Copy number variations present in tumors, as well as tumor-specific mutations were faithfully reflected in meningioma EV-DNA. Proteomic EV profiling did not permit original tumor identification but revealed tumor-associated proteins such as desmoplakin that could potentially be utilized to enrich meningioma EVs from biofluids. In conclusion, elevated EV levels in meningioma patient plasma may aid in tumor diagnosis and assessment of treatment response. Meningioma EV-DNA mirrors genetic and epigenetic tumor alterations and facilitates molecular tumor classification.