Extracellular Vesicles Concentration Research Articles

Urinary extracellular vesicle dynamics in Parkinson's disease patients with urinary dysfunction.

Parkinson's disease (PD) presents with severe motor manifestations and a plethora of non-motor symptoms. Urinary dysfunctions are one of the most common non-motor symptoms of PD patients responsible for reduced quality of life. Urinary extracellular vesicles (EVs) are mostly considered to originate from the cells in the urogenital tract. In this study, we have performed urinary EV analysis in 29 PD cases with varied severity of urinary dysfunction and correlated it with the EV dynamics in 29 age-matched controls. In the studied cases, apart from urinary dysfunction, symptoms of depression, anxiety, cognitive dysfunction, sleep, and wakefulness were observed in >75% of the cases. No significant difference in urinary EV size, concentration and urinary EV protein concentration was observed between PD cases with urinary dysfunction and controls. However, a significant positive association was observed between urinary EV concentration and motor scores (p = 0.042), while no association was observed between urinary EV concentration and urinary dysfunction scores. Chronic stress induced by motor symptoms could be one of the reasons for excessive EV production in PD patients with urinary dysfunctions. Large-scale studies on the association of urinary EV dynamics with motor and non-motor symptoms may provide additional information on urinary dysfunction in PD.

Erythrocyte-derived extracellular vesicles from type 2 diabetes patients induce endothelial dysfunction through arginase 1

Abstract Background The mechanisms driving the development of cardiovascular injury in type 2 diabetes (T2D) remain incompletely understood. We recently demonstrated that red blood cells (RBCs) from patients with T2D (T2D-RBCs) act as mediators of endothelial dysfunction, but the mechanisms underlying this interaction are not clarified. We found that RBCs via upregulation of arginase 1 attenuate nitric oxide bioavailability and endothelial function. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by practically all cell types, including RBCs, and represent a novel mechanism of intercellular communication. Purpose This study aimed to determine whether EVs derived from T2D-RBCs are involved as mediators in vascular injury through the signalling of arginase 1. Methods T2D-RBCs and RBCs from age-matched healthy controls (H-RBCs) were isolated and incubated with Krebs-Henseleit buffer (20% haematocrit). Following 18h incubation, the conditioned medium was collected for EV isolation using sequential ultracentrifugation and membrane affinity column. EV concentration was measured by nanoparticle tracking analysis. Aortas isolated from wild-type mice were incubated with EVs derived from T2D-RBCs and H-RBCs for 18h. Endothelium-dependent and -independent relaxations (EDR and EIDR, respectively) of the aortae were evaluated in a wire myograph. The involvement of arginase was investigated by the addition of the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) either to the 18h co-incubation of EVs with the aortic segments to selectively investigate the contribution of EV-derived arginase (ABH, 10 mM) or to the aortae following the 18h co-incubation to selectively target vascular arginase (ABH, 100 µM). All animal experiments were performed according to the principles of laboratory animal care (NIH Publication no. 85-23 revised 1985) and human procedures according to the declaration of Helsinki and approved by the Swedish Ethical Review Authority. Results T2D-RBCs had a ten times lower EV concentration compared to H-RBCs. T2D-RBC-derived EVs isolated using both methods significantly impaired EDR (Fig. 1A-C), whereas EIDR was not affected. This effect was observed irrespective of if the same volume or concentration of EVs were administered. Inhibition of arginase with ABH during the co-incubation with EVs derived from T2D-RBCs completely prevented the impairment of EDR induced by the EVs (Fig. 1D). Administration of ABH to the aortae following the co-incubation also attenuated the impairment of EDR. Immunohistochemical staining revealed upregulation of arginase 1 (Fig. 2A-B) but not arginase 2 (Fig. 2C-D) in the vasculature following incubation with EVs from T2D-RBCs. Conclusion EVs derived from T2D-RBCs induce endothelial dysfunction, and arginase 1 derived from these EVs mediates this endothelial dysfunction. These results shed new important light on the mechanism underlying vascular injury mediated by RBCs in T2D.

Abstract 13999: The Relationship Between Neighborhood Deprivation, Diabetes, and Extracellular Vesicles in African American Women at Risk for CVD: Data From a Pilot Study of the Step It Up Physical Activity Intervention

Background: African American women (AA) are at disproportionally high risk for diabetes and cardiovascular disease (CVD) which is accelerated when residing in resource-limited neighborhoods. Extracellular vesicles (EV) are emerging to be of importance in CVD development and progression, but little is known about a potential impact of neighborhood socioeconomic deprivation (NSD). In this study we examined associations between NSD, plasma EV, and markers of diabetes risk in a community-based cohort of AA women at risk for CVD from resource-limited neighborhoods in Washington, DC. Methods: Participants were enrolled in pilot testing of Step It Up, a community engaged physical activity intervention. Blood samples were drawn to measure clinical labs during participants’ baseline visit to the NIH Clinical Center. EV were isolated from heparinized plasma using size exclusion chromatography with size and numbers determined by nanoparticle tracking analysis. NSD was calculated using 2018 US Census tract data as a measure of neighborhood deprivation. Associations between NSD, EV, and diabetes-related clinical measures were analyzed using multivariable regressions adjusted for BMI and ASCVD 10-year risk score. Results: The study cohort was a convenience sample of AA women at risk for CVD (N=24, Age: 57±12, BMI: 35±6, ASCVD: 9±5). NSD associated with fasting glucose (β=0.54, p=0.007) and Hemoglobin A1c (HA1c) (β=0.51, p=0.007). Additionally, NSD associated with lower EV size (β=-0.42, p=0.03), with no association with EV concentration. Fasting glucose and HA1c associated negatively with EV size (β=-0.48, p=0.03 and β=-0.50, p=0.02, respectively) but no association was found with EV concentration. Conclusions: Thus, our data show that higher neighborhood deprivation is associated with lower EV size which, in turn, is associated with higher diabetes risk markers. More research is needed to understand the role of EV in diabetes and CVD development and progression. While larger studies further evaluating these observations are needed, our data highlight the importance of increased diabetes screening and multi-level interventions in patients residing in under-resourced communities to reduce existing health disparities.

Abstract 17627: The Relationship Between Physical Activity, Extracellular Vesicles, and Diabetes Risk in African American Women at Risk for Cardiovascular Disease: Data From a Pilot Study of the Step It Up Physical Activity Intervention

Background: Diabetes and cardiovascular disease (CVD) disproportionally affect African American (AA) women. Prior studies suggest extracellular vesicles (EV) are involved in atherogenesis, but little is known about relationships between PA and EVs in AA women. Thus, we examined associations between diabetes risk markers, EV, and PA in a community-based cohort of AA women at risk for CVD from resource-limited Washington, D.C. neighborhoods. Methods: Participants were enrolled in pilot testing of Step It Up, a place-tailored mobile app designed to increase PA. Baseline daily step counts for PA were measured by Fitbit® (Charge 2) and fasting baseline blood samples were cryopreserved. NMR (Nuclear magnetic resonance) spectroscopy was used to measure Lipoprotein Insulin Resistance Index (LP-IR), a diabetes risk marker. EV were isolated from heparinized plasma using size exclusion chromatography with size and numbers determined by nanoparticle tracking analysis. Associations between LP-IR, EVs, and daily step count were analyzed using multivariable regressions adjusted for BMI and ASCVD 10-year risk score. Results: The study cohort was a convenience sample of AA women with overweight/obesity (N=24, Age: 57±12, BMI: 35±6, ASCVD: 9±5). Smaller EV size associated with higher LP-IR (β=-0.45, p=0.04) with EV concentration trending to a positive association with LP-IR (β=0.38, p=0.08). Higher daily step count associated with larger EV particle size (β=0.48, p=0.02) but not with EV concentration. Additionally, higher daily step count associated with lower LP-IR (β=-0.42, p=0.04). Conclusions: Our data show that reduced EV size and increased EV concentration associate with higher LP-IR, a diabetes index among a community-based cohort of AA women. Additionally, our findings suggest that PA might help mitigate these associations. More research is needed to understand the potential impact of PA on EV, diabetes risk and subsequent CVD. Future PA interventions in at-risk patients may reduce existing diabetes and CVD-related health disparities and EVs may emerge as a mechanistic link.

Abstract 16961: Characterization of Extracellular Vesicles and RNA Content and Its Relevance to Thrombo-Inflammatory Biomarkers in Patients With Acute Pulmonary Embolism

Introduction: Pulmonary embolism (PE) is a complex disease with significant morbidity and mortality rates globally. Previous studies have shown that extracellular vesicles (EV) carrying disease specific ribonucleic acids (RNA’s) may contribute to pro-inflammatory and pro-coagulant state in VTE. This study aimed on profiling EV related parameters, RNA content, and thrombo-inflammatory markers in patients with acute PE compared to healthy individuals. Materials & Methods: Prospective cohort of patients with PE (n=72) were compared with normal healthy individuals (n=19) and further stratified by PE severity. Blood samples were collected under an IRB-approved project from Loyola University Chicago. Plasma samples were analyzed for the EV parameters, RNA parameters, and thrombo-inflammatory markers (D-Dimer, PAI-1, and CRP) respectively. Results: PE cohort includes 72 patients (41 male, and 31 female) with a median age of 67-years (interquartile range; IQR= 53-76), and a median body mass index (BMI) of 27.5-kg/m 2 (IQR= 24.7-35.7). Compared to controls, EV concentration, and EV/ng RNA were significantly (p= <0.05) lower, while D-Dimer, CRP and PAI-1 were significantly (p= <0.05) higher in patients with PE. In correlation analysis, varying levels of positive and negative correlations between the EV, and RNA parameters, and thrombo-inflammatory markers were noted (Figure 1). Upon stratification on PE severity, D-dimer was higher in massive PE compared to low-risk (p= 0.012), and sub-massive PE (p=0.036) respectively. Conclusion: These studies demonstrate significant changes in extracellular vesicles, RNA content and thrombo-inflammatory markers in acute PE patients compared to healthy controls. These changes may be due to the drivers of VTE pathogenesis or the result of a consumption process in acute PE. Further large scales studies on disease specific exosomes and RNA signatures are warranted to understand their relevance.

MicroRNAs in extracellular vesicles released from epicardial adipose tissue promote arrhythmogenic conduction slowing

Patients with excess epicardial adipose tissue (EAT) are at increased risk of developing cardiac arrhythmias. EAT promotes arrhythmias by depolarizing the resting membrane of cardiomyocytes, which slows down conduction and facilitates re-entrant arrhythmias. We hypothesized that EAT slows conduction by secreting extracellular vesicles (EVs) and their microRNA (miRNA) cargo. We aimed to determine the role of EAT-derived EVs and their miRNA cargo in conduction slowing. EAT and subcutaneous adipose tissue (SAT) were collected from patients with atrial fibrillation. Adipose tissue explants were incubated in culture medium and secretome was collected. The numbers of EVs in the EAT and SAT secretome were measured by calibrated flow cytometry. EVs in the EAT secretome were isolated by size exclusion chromatography and miRNAs were sequenced. Pathway analysis was performed to predict candidates involved in cardiac electrophysiology. The candidates were validated in the EAT and SAT by quantitative real-time polymerase chain reaction. Finally, miRNA candidates were overexpressed in neonatal rat ventricular myocytes. The EV concentration was higher in the EAT secretome than in the SAT and control secretomes. miRNA sequencing of EAT-derived EVs detected a total of 824 miRNAs. Pathway analysis led to the identification of 7 miRNAs potentially involved in regulation of cardiac resting membrane potential. Validation of those miRNA candidates showed that they were all expressed in EAT, and that miR-1-3p and miR-133a-3p were upregulated in EAT in comparison with SAT. Overexpression of miR-1-3p and miR-133a-3p in neonatal rat ventricular myocytes led to conduction slowing and reduced Kcnj2 and Kcnj12 expression. miR-1-3p and miR-133a-3p are potential mediators of EAT arrhythmogenicity.

Efficacy of Mesenchymal-Stromal-Cell-Derived Extracellular Vesicles in Ameliorating Cisplatin Nephrotoxicity, as Modeled Using Three-Dimensional, Gravity-Driven, Two-Layer Tubule-on-a-Chip (3D-MOTIVE Chip).

Mesenchymal stromal cell (MSC)-derived extracellular vesicles (EVs) are known to have a therapeutic effect on nephrotoxicity. As animal models require significant time and resources to evaluate drug effects, there is a need for a new experimental technique that can accurately predict drug effects in humans. We evaluated the therapeutic effect of MSC-derived EVs in cisplatin nephrotoxicity using a three-dimensional, gravity-driven, two-layer tubule-on-a-chip (3D-MOTIVE chip). In the 3D-MOTIVE chip, 10 μM cisplatin decreased the number of attached cells compared to the vehicle. Conversely, annexin V and reactive oxygen species (ROS) were increased. Cell viability was increased 2.8-fold and 2.5-fold after treatment with EVs at 4 and 8 µg/mL, respectively, compared to the cisplatin-induced nephrotoxicity group. Cell attachment was increased 2.25-fold by treatment with 4 µg/mL EVs and 2.02-fold by 8 µg/mL EVs. Annexin V and ROS levels were decreased compared to those in the cisplatin-induced nephrotoxicity group. There were no significant differences in annexin V and ROS levels according to EV concentration. In sum, we created a cisplatin-induced nephrotoxicity model on a 3D-MOTIVE chip and found that MSC-derived EVs could restore cell viability. Thus, MSC-derived EVs may have the potential to ameliorate cisplatin-induced nephrotoxicity.

Influence of the Anesthetic Technique on Circulating Extracellular Vesicles in Bladder Cancer Patients Undergoing Radical Cystectomy: A Prospective, Randomized Trial.

Anesthetics have been shown to alter tumor progression and seem to influence surgical cancer outcome. Circulating extracellular vesicles as mediators of intercellular communication are involved in cancer progression and may be influenced by anesthetics. In this prospective, randomized study, effects of anesthetics on extracellular vesicles and associated micro-RNAs in bladder cancer patients undergoing radical cystectomy were tested. Extracellular vesicles from 51 patients at four perioperative time points receiving Propofol or Sevoflurane were extracted with polymer-based methods and quantified with a nanoparticle-tracking analysis. Vesicle-associated micro-RNAs were analyzed with a real-time polymerase chain reaction using array cards and single assays for tumor-associated miR-21-5p, miR-15a-5p, miR-17-5p and miR-451a. Plasma extracellular vesicle concentration (suture: fold change (fc) in Propofol at 4.1 ± 3.9 vs. Sevoflurane at 0.8 ± 0.5; p = 0.003) and associated miRNAs increased significantly (+30% post induction, +9% 30 Min surgery) in the Propofol group. Tumor-associated miRNAs increased during surgery in both groups (fc in miR-21-5p: 24.3 ± 10.2, p = 0.029; fc in miR-15a-5p: 9.7 ± 3.8, p = 0.027; fc in miR-17-5p: 5.4 ± 1.7, p = 0.014), whereas antitumor miR-451a increased in the Propofol group only (fc: 2.5 ± 0.6 vs. 1.0 ± 0.2; p = 0.022). Anesthetics influence extracellular vesicles and associated micro-RNAs of bladder cancer patients during surgery. Increased expression of antitumor micro-RNA may be an explanatory approach for decreased tumor cell viability after Propofol.

Homogenization and thermal processing reduce the concentration of extracellular vesicles in bovine milk.

Extracellular vesicles (EVs) in bovine milk confer beneficial physiologic effects to consumers. Industrial processing treatments may affect the amount or bioactivity of EVs intrinsic to bovine milk. We investigated how the content and concentration of EVs were affected by homogenization and thermal processing of raw bovine milk. Raw milk was processed by homogenization, low-temperature (LT) heat, or pasteurization [high-temperature short-time (HTST) and ultra-high-temperature (UHT)] in a pilot processing facility. EVs were isolated from the raw and processed bovine milk using differential ultracentrifugation and quantified using a nanoparticle tracking analyzer. Bovine milk EVs were assessed for total miRNA and protein concentrations standardized to particle count using a fluorometric assay. There were 1.01 × 1010 (±3.30 × 109) EV particles per ml of bovine milk. All industrial processing treatments caused >60% decrease in EV concentration compared to the raw bovine milk. Homogenization and heat treatments independently and additively reduced the content of EVs in bovine milk. The averages of total miRNA/particle and total protein/particle concentrations were elevated threefold by low-temperature heat-processing treatment relative to HTST and UHT pasteurizations. The average diameter of EVs was reduced by 11%-16% by low temperature compared to raw milk (127 ± 13 nm). Homogenization and pasteurization indiscriminately reduce the EV concentration of bovine milk. Smaller EVs with higher protein content resist degradation when processing bovine milk at sub-pasteurization temperature. This new foundational knowledge may contribute to food product development on the preservation of EVs in processed dairy products, including bovine milk-based infant formulas that some newborns are dependent on for adequate growth and development.

Umbilical cord mesenchymal stromal cell-derived extracellular vesicles lack the potency to immunomodulate human monocyte-derived macrophages in vitro

Mesenchymal stromal cells (MSCs) have been reported to display efficacy in a variety of preclinical models, but without long-term engraftment, suggesting a role for secreted factors, such as MSC-derived extracellular vesicles (EVs). MSCs are known to elicit immunomodulatory effects, an important aspect of which is their ability to affect macrophage phenotype. However, it is not clear if these effects are mediated by MSC-derived EVs, or other factors secreted by the MSCs. Here, we use flow cytometry to assess the effects of human umbilical cord (hUC) MSC-derived EVs on the expression of pro-inflammatory (CD80) and anti-inflammatory (CD163) surface markers in human monocyte-derived macrophages (hMDMs). hUC-MSC-derived EVs did not change the surface marker expression of the hMDMs. In contrast, when hMDMs were co-incubated with hUC-MSCs in indirect co-cultures, changes were observed in the expression of CD14, CD80 and CD163, particularly in M1 macrophages, suggesting that soluble factors are necessary to elicit a shift in phenotype. However, even though EVs did not alter the surface marker expression of macrophages, they promoted angiogenesis and phagocytic capacity increased proportionally to increases in EV concentration. Taken together, these results suggest that hUC-MSC-derived EVs are not sufficient to alter macrophage phenotype and that additional MSC-derived factors are needed.

MicroRNA signature from extracellular vesicles of HCV/HIV co-infected individuals differs from HCV mono-infected

Hepatitis C virus (HCV) coinfection with human immunodeficiency virus (HIV) has a detrimental impact on disease progression. Increasing evidence points to extracellular vesicles (EVs) as important players of the host-viral cross-talk. The microRNAs (miRNAs), as essential components of EVs cargo, are key regulators of normal cellular processes and also promote viral replication, viral pathogenesis, and disease progression. We aimed to characterize the plasma-derived EVs miRNA signature of chronic HCV infected and HIV coinfected patients to unravel the molecular mechanisms of coinfection. EVs were purified and characterized from 50 plasma samples (21 HCV mono- and 29 HCV/HIV co-infected). EV-derived small RNAs were isolated and analyzed by massive sequencing. Known and de novo miRNAs were identified with miRDeep2. Significant differentially expressed (SDE) miRNA identification was performed with generalized linear models and their putative dysregulated biological pathways were evaluated. Study groups were similar for most clinical and epidemiological characteristics. No differences were observed in EVs size or concentration between groups. Therefore, HCV/HIV co-infection condition did not affect the concentration or size of EVs but produced a disturbance in plasma-derived EVs miRNA cargo. Thus, a total of 149 miRNAs were identified (143 known and 6 de novo) leading to 37 SDE miRNAs of which 15 were upregulated and 22 downregulated in HCV/HIV co-infected patients. SDE miRNAs regulate genes involved in inflammation, fibrosis, and cancer, modulating different biological pathways related to HCV and HIV pathogenesis. These findings may help to develop new generation biomarkers and treatment strategies, in addition to elucidate the mechanisms underlying virus–host interaction.Key messagesHCV and HCV/HIV displayed similar plasma-EV size and concentration.EVs- derived miRNA profile was characterized by NGS.37 SDE miRNAs between HCV and HCV/HIV were observed.SDE miRNAs regulate genes involved in inflammation, fibrosis and cancer.

One-week storage of refrigerated bovine milk does not affect the size, concentration, or molecular properties of extracellular vesicles

Milk extracellular vesicles (EVs) have gained extensive attention as promising diagnostic and therapeutic tools. Pre-analytical raw milk storage at low temperatures is an ordinary and usually necessary step after sample collection. It is known that direct freezing of unprocessed whole milk contaminates the native pool of milk EVs with other cell structures. However, less evidence is available regarding prolonged cooling at 4°C. The current study assessed whether pre-analytical storage of bovine raw milk for several days affected EV isolation and further analysis. To confirm the independence from the health status of the mammary gland, we analyzed milk samples stored at 4°C for 1, 2, 3, and 7 d past collection, respectively, from 2-quarters of the same cow with different somatic cell counts (SCC). Seven days of refrigeration did not change the milk EVs' size, concentration, or morphology. We neither detected changes in the EV cargo regarding the amount of protein and RNA nor the specific EV markers TSG101, CD9, and CD81 in milk from quarters with high and low somatic cell counts. Overall, we observed fewer CD81 and CD9 markers in quarters with a high SCC. Moreover, there was no reduction in the mastitis-related miRNA bta-miR-223-3p, suggesting that refrigeration for several days up to one week is a possible storage option compatible with further EV analyses. The findings of this study enhance the confidence that milk EVs are highly stable in the raw milk matrix.

Comparative Studies of Different Preservation Methods and Relative Freeze-Drying Formulations for Extracellular Vesicle Pharmaceutical Applications.

Extracellular vesicles (EVs) have been studied for years for their role as effectors and mediators of cell-to-cell communication and their potential application to develop new and increasingly performing nanotechnological systems for the diagnosis and/or treatment of many diseases. Given all the EVs applications as just isolated, functionalized, or even engineered cellular-derived pharmaceuticals, the standardization of reliable and reproducible methods for their preservation is urgently needed. In this study, we isolated EVs from a healthy blood cell line, B lymphocytes, and compared the effectiveness of different storage methods and relative freeze-drying formulations to preserve some of the most important EVs' key features, i.e., concentration, mean size, protein content, and surface antigen's expression. To develop a preservation method that minimally affects the EVs' integrity and functionality, we applied the freeze-drying process in combination with different excipients. Since EVs are isolated not only from body fluids but also from culture media conditioned by the cells growing there, we decided to test both the effects of the traditional pharmaceutical excipient and of biological media to develop EVs solidified products with desirable appearance and performance properties. Results showed that some of the tested excipients, i.e., sugars in combination with dextran and glycine, successfully maintained the stability and integrity of EVs upon lyophilization. In addition, to evaluate the preservation of the EVs' biological activity, we assessed the cytotoxicity and internalization ability of the reconstituted EVs in healthy (B lymphocytes) and tumoral (Burkitt's lymphoma) cells. Reconstituted EVs demonstrated toxicity only toward the cancerous cells, opening new therapeutic opportunities for the oncological field. Furthermore, our study showed how some biological or cellular-conditioned fluids, commonly used in the field of cell cultures, can act not only as cryoprotectants but also as active pharmaceutical ingredients, significantly tuning the therapeutic effect of EVs, even increasing their cellular internalization.

Exploration of Extracellular Vesicle Long Non-Coding RNAs in Serum of Patients with Cervical Cancer

Introduction: Cervical cancer (CC) is the fourth most common cancer type and a leading cause of cancer-related deaths in women worldwide. Its underlying molecular mechanisms are unclear. Cancer cell-derived extracellular vesicles (EVs) are involved in cancer development and progression by delivering regulatory factors, including microRNAs and long non-coding RNAs (lncRNAs). Methods: Here, we identified the EV lncRNA expression profiles associated with different developmental stages of CC using next-generation sequencing. EVs from the serum of patients with stages I–III CC and healthy donors were characterized using EV marker immunoblotting and transmission electron microscopy. Results: The EV concentration increases with progression of the disease. Most particles had a 100–250-nm diameter, and their sizes were similar in all groups. We identified many lncRNAs that were uniquely and differentially expressed (DE) in patients with different stages of CC. The pathway analysis results indicated that the upregulated DE EV lncRNAs abundant in stages I and II were associated with cell proliferation and inflammation and cancer progression pathways, respectively. LINC00941, LINC01910, LINC02454, and DSG2-AS1 were highly expressed, suggesting poor overall survival of CC patients. Interestingly, DSG2-AS1 was associated with the human papillomavirus infection pathway through AKT3, DLG1, and COL6A2 genes. Conclusion: This is the first study that reports the levels of EVs and their lncRNA contents change during cancer development, demonstrating the existence of a unique vesicle-mediated cell-to-cell communication network underlying cancer progression.

Quantitative increases of extracellular vesicles in prolonged cold storage of platelets increases the potential to enhance fibrin clot formation.

Platelet derived extracellular vesicles (EVs) display a pro-coagulant phenotype and are generated throughout platelet concentrate (PC) storage. Cold storage (CS) of PCs is thought to provide a superior haemostatic advantage over room temperature (RT) storage and could prolong the storage time. However, the effect of storage conditions on EV generation and PC function is unknown. We investigated EV production under CS and RT conditions and assessed whether these EVs exhibited a more pro-coagulant phenotype in model experiments. Buffy-coat-derived PCs in a platelet additive solution (PAS) to plasma ratio of approximately 65:35 were stored at RT (22 ± 2°C) or CS (4 ± 2°C) for a prolonged storage duration of 20 days. Impedance aggregometry assessed platelet function. EVs were isolated throughout storage and quantified using nanoparticle tracking analysis. EVs were applied to a coagulation assay to assess the impact on fibrin clot formation and lysis. CS produced significantly larger EVs from day 4 onwards. EV concentration was significantly increased in CS compared to RT from day 15. EVs, regardless of storage, significantly reduced time to clot formation and maximum optical density measured compared to the no EV control. Clot formation was proportionate to the number of EV applied but was not statistically different across storage conditions when corrected for EV number. EVs in CS and RT units showed similar clot formation capacity. However, the higher number of larger EVs generated in CS compared to RT suggests PC units derived from CS conditions may overall exhibit a haemostatically superior capacity compared to RT storage.

Abstract P2179: Endothelial-derived Extracellular Vesicles Communicate With Cardiomyocytes To Elicit Cancer Therapy-related Cardiac Dysfunction

Background: Treatment with the anthracycline, epirubicin, can result in cancer-therapy related cardiac dysfunction (CTRCD), a leading cause of non-malignant mortality in cancer survivors. Endothelial cells (ECs) lining the blood vessels are the first point of contact of epirubicin and secrete factors that are pivotal in regulating cardiac function. Of these factors, extracellular vesicles (EVs) are known to mediate intercellular communication. It remains unknown whether and how EC-EVs affect cardiomyocyte function in the context of CTRCD. Hypothesis: We hypothesize that epirubicin exposure leads to altered EC-EV cargo which mediates cardiomyocyte dysfunction. Methods: Human umbilical vein endothelial cells (HUVEC) were exposed to the peak and trough concentrations of epirubicin and endothelial function was assessed. EC-EVs were isolated via ultracentrifugation and EV size and concentration were measured through nanoparticle tracking analysis. The protein content of EC-EVs was determined using liquid chromatography with tandem mass spectrometry. Pluripotent stem cell-derived cardiomyocytes (iPSC-CM) were exposed to physiological concentrations of EVs (1x1010 particles/mL) and contractility was assessed in real-time. Results: Epirubicin exposure increased endothelial leak, apoptosis, and adhesion molecule expression. We observed an increase in size and concentration of EC-EVs and proteomic analysis revealed altered EV cargo including reduced cardiomyocyte protective factors, CNP and eNOS in EVs from epirubicin treated HUVEC in comparison to control. Treatment of iPSC-cardiomyocytes with EVs from epirubicin treated HUVECs resulted in reduced contractility compared to EVs from healthy ECs. Cleavage of EC-EV surface proteins via proteinase K abrogated the effects of EC-EVs on iPSC-cardiomyocytes. Cardiomyocyte-specific pathways targeted by EC-EVs from epirubicin- compared to control-treated cells are currently on-going. Conclusion: We demonstrate that epirubicin treatment leads to endothelial dysfunction and altered release of EVs capable of mediating cardiomyocyte dysfunction. Our results show the potential role of EC-EVs in CTRCD pathogenesis and highlight their influence on cardiomyocyte function.

Abstract P3158: Extracellular Vesicles From Ischemic Myocardium Mediate Regulatory T Cell Dysfunction Through MicroRNA-1-mediated Suppression Of VEGF-A

Motivation: Myocardial infarction (MI) triggers an efflux of pro-inflammatory extracellular vesicles (EVs) from the heart into the circulation. Here, we test the hypothesis that circulating ischemic heart EVs (iEVs) attenuate regulatory T cell (Treg) proliferation and function post-MI. Methods: Hearts were explanted from mice and Langendorff-perfused with Tyrode buffer either continuously for two hours (nonischemic), or subjected to no-flow for 30 min, and reperfused for 1.5h. Heart effluent (~700ml) was collected for EV isolation by ultracentrifugation. EV size and concentration were measured by nanoparticle tracking analysis; EV markers were characterized by western blot. EVs from nonischemic heart effluent (nEVs) served as controls. RNA sequencing (RNAseq) followed by qPCR validation was used to identify microRNAs in EVs. Assessments of human Tregs included cell proliferation (Cell Counting Kit-8 assay), IL-10 production (qPCR of Tregs, and ELISA of Treg-conditioned media), and RNAseq after exposure to EVs. In vivo, an antagomir was administered retro-orbitally immediately post-MI, and splenic Tregs were measured by flow cytometry 7 days post-MI. Findings: MiR-1-3p was highly enriched in iEVs compared to nEVs. Exposure of human Tregs to iEVs or a miR-1 mimic resulted in decreased proliferation ( P <0.0001, N=8) and production of IL-10 (qPCR: P<0.0001, n=9; ELISA: P <0.0001, N=6) compared to nEVs. Antagomir-1 reversed iEV- and miR-1-induced inhibition of Treg proliferation. RNAseq on human Tregs revealed that VEGF-A, a miR-1 target gene, was suppressed after exposure to iEVs compared to nEVs. In vivo, the number of splenic Tregs decreased on day 7 post-MI, with a concomitant decrease in IL-10 + and VEGF-A + Tregs. Intriguingly, administration of antagomiR-1 restored numbers of splenic IL10 + and VEGF-A + Treg cells. Conclusions: iEVs cause Treg dysfunction by transferring miR-1. In turn, miR-1 suppresses VEGF-A, a known modulator of Treg function. Our findings reveal novel paracrine modulation of immune cells by ischemic myocardium. Targeting EV signaling post-MI represents a potential new strategy to augment Treg-mediated tissue repair.

Abstract P3151: A Unique Profile Of Extracellular Vesicles In Marfan Patients With Thoracic Aortic Aneurysm

Objectives: Extracellular vesicles (EVs) [exosomes and microvesicles] have significant diagnostic and therapeutic potential, but clinical application is limited by disparate methods of data collection. Exosomes are 30-100nm, and microvesicles are 100-300nm lipid bilayer vesicles that originate from all cell types. Marfan Syndrome (MFS) is a connective tissue disorder. Thoracic aortic aneurysm (TAA) formation and dissection are the leading causes of death in MFS patients. Early identification of TAAs in MFS patients is imperative and will mitigate morbidity and mortality. This study’s goal was to develop a reproducible workflow for isolation and quantification of EVs 30-300nm and test the hypothesis that number and size distribution are altered in MFS patients with TAA. Methods: A method of standardized reproducible measurement of EVs isolated from plasma was developed. This includes isolation by automated size exclusion chromatography and quantification of size distribution by tunable resistive pulse sensing. Characteristic markers specific to both EV subtypes were confirmed by immunoblot. Results: EVs isolated from control plasma (n=12) and Marfan TAA plasma (n=6) were measured for concentration (control 0.60 x 10 11 particles/mL, MFS TAA 1.47 x 10 11 particles/mL), mean diameter (control 77.75nm, MFS TAA 69.92nm), mode diameter (control 65.50nm, MFS TAA 60.50nm), and d90/d10 ratio (control 1.68, MFS TAA 271 1.57). Conclusion: MFS patients with TAA have more EVs with a smaller diameter, suggesting increased export and a shift in ratio of microvesicles:exosomes. This may be used as a diagnostic tool or potential therapeutic target. This scalable protocol ensures recovery of intact EVs suitable for downstream biochemical and functional analyses. Simultaneous measurement quantifies EV concentration and size distribution absolutely, correcting for variations, providing a novel method of standardization.

Characterization of extracellular vesicles in COVID-19 infection during pregnancy.

Introduction: SARS-CoV-2 infection may cause a severe inflammatory response, inflicting severe morbidity and mortality. This risk is modestly increased in pregnant patients. Despite the hypercoagulability and immunosuppression associated with pregnancy, most pregnant women experience a mild COVID-19 infection. Maternal extracellular vesicles (EVs) may interact with endothelial and immune components to facilitate a favorable disease course. This pilot study aimed to explore the characteristics of EVs released during COVID-19 infection occurring during the third trimester of pregnancy. Methods: In this prospective study, blood samples were obtained from 16 healthy non-pregnant (NP), 18 healthy-pregnant (HP), and 22 COVID-19 positive pregnant subjects (CoV-P). Disease course and pregnancy outcomes were assessed and EVs were characterized. Of note, limited volumes of sample acquired from the subjects made it necessary to use smaller and different subsets of samples for each analysis. Results: The majority (91%) of the COVID-19-pregnant subjects (18 mild and 2 moderate disease) experienced good pregnancy-related outcomes. EV concentrations were higher in healthy-pregnant subjects compared to non-pregnant subjects (p = 0.0041) and lower in COVID-19-pregnant subjects compared to healthy-pregnant subjects (p = 0.0150). CD63 exosome marker expression was higher in EVs of healthy-pregnant subjects and COVID-19-pregnant subjects compared to EVs of non-pregnant subjects (p = 0.0149, p = 0.0028, respectively). Similar levels of SARS-CoV-2 entry proteins (ACE-2 and TMPRSS2) were found in all three groups. Cytokine content increased in healthy-pregnant subject-EVs compared to non-pregnant EVs, while IL-2 and IL-6 levels were decreased in COVID-19-pregnant subject-EVs compared to healthy-pregnant subject-EVs (p = 0.043, p = 0.0390, respectively). CD8+, cytotoxic T-cell marker, was lower in non-pregnant EVs compared to healthy-pregnant subject-EVs and to COVID-19-pregnant subjects (p = 0.0108, p < 0.0001, respectively). COVID-19- pregnant subject-EVs demonstrated higher levels of platelet activation marker (CD62P) than non-pregnant (p = 0.0327) and healthy-pregnant subjects (p = 0.0365). Endothelial marker EV-CD144+ was lower in healthy-pregnant subjects versus non-pregnant subjects (p = 0.0093), but similar in COVID-19-pregnant and non-pregnant subjects. Other EVs' coagulation markers/activity, D-Dimer and fibrinogen levels were similar in healthy-pregnant subjects and COVID-19 positive pregnant subjects. Conclusion: COVID-19 positive pregnant subjects' EVs demonstrated an attenuated inflammatory response, with no additional activation of the coagulation system.

Extracellular vesicles in porcine seminal plasma maintain sperm function by reducing Lyso-PC

Seminal plasma (SP) exosomes are membrane vesicles with 30−120 nm diameter, which have been shown to regulate the physiological processes in sperm cells. However, the mechanism through which SP extracellular vesicles (EVs) maintain sperm function remains unclear. To explore the mechanism of EVs in sperm preservation, different EV concentrations (EV-0, EV-1, EV-4, EV-8, EV-16) were added to semen liquid preservation solution. Sperm motility was detected via computer-assisted sperm analysis systems, plasma membrane integrity was assessed using SYBR-14 and propidium iodide staining, and total antioxidant capacity and lipid oxidation levels were evaluated after 0, 2, 4, 6 days of liquid-storage at 17 °C. Differences in sperm metabolites between the EV-0 and EV-8 groups stored for 4 days, were analyzed using untargeted metabolomics [liquid chromatography with tandem mass spectrometry (LC-MS/MS)]. Moreover, mitochondrial reactive oxygen species (mtROS) levels were assessed when sperm incubated with EV-0 and EV-8 during 0, 2, 4, 6 days, and embryonic development were assessed when sperm incubated with EV-0 and EV-8 stored for 4 days at 17 °C. Our data showed that compared to EV-0, sperm motility and plasma membrane integrity, antioxidant capacity, lipid oxidation levels and cleavage rates were significantly increased in the EV-8 group (P < 0.05), lysophosphatidylcholine (Lyso-PC) metabolisma and mtROS levels were significantly reduced in the EV-8 group (P < 0.05). In conclusion, SP EVs inhibit the premature increase in Lyso-PC, exert antioxidant effects on mtROS, and preserve sperm's ability to fuse with oocytes.