Levels Of Microvesicles Research Articles

Bilberry Supplementation after Myocardial Infarction Decreases Microvesicles in Blood and Affects Endothelial Vesiculation.

ScopeDiet rich in bilberries is considered cardioprotective, but the mechanisms of action are poorly understood. Cardiovascular disease is characterized by increased proatherogenic status and high levels of circulating microvesicles (MVs). In an open‐label study patients with myocardial infarction receive an 8 week dietary supplementation with bilberry extract (BE). The effect of BE on patient MV levels and its influence on endothelial vesiculation in vitro is investigated.Methods and resultsMVs are captured with acoustic trapping and platelet‐derived MVs (PMVs), as well as endothelial‐derived MVs (EMVs) are quantified with flow cytometry. The in vitro effect of BE on endothelial extracellular vesicle (EV) release is examined using endothelial cells and calcein staining. The mechanisms of BE influence on vesiculation pathways are studied by Western blot and qRT‐PCR. Supplementation with BE decreased both PMVs and EMVs. Furthermore, BE reduced endothelial EV release, Akt phosphorylation, and vesiculation‐related gene transcription. It also protects the cells from P2X7‐induced EV release and increase in vesiculation‐related gene expression.ConclusionBE supplementation improves the MV profile in patient blood and reduces endothelial vesiculation through several molecular mechanisms related to the P2X7 receptor. The findings provide new insight into the cardioprotective effects of bilberries.

High levels of endothelial and platelet microvesicles in patients with type 1 diabetes irrespective of microvascular complications

IntroductionPatients with type 1 diabetes have high risk of developing microvascular complications, and microangiopathy contributes to premature cardiovascular disease in this population. The role that microvesicles (MVs) may play in the development of microangiopathy in type 1 diabetes remains unclear. Materials and methodsPlasma levels of endothelial MVs (EMVs) and platelet MVs (PMVs) in 130 patients with type 1 diabetes without microangiopathy, 106 patients with microangiopathy and 100 matched healthy controls were analyzed using flow cytometry. MV expression of procoagulant phosphatidylserine (PS) and proinflammatory high mobility group box-1 protein (HMGB1) was also assessed. ResultsPatients with type 1 diabetes had markedly elevated levels of EMVs and PS+ EMVs as well as PMVs and PS+ PMVs compared to healthy controls (p < .001 for all). Furthermore, HMGB1+ EMVs and HMGB1+ PMVs were significantly increased in patients (p < .001 for all). After adjusting for potential confounders, there were no clear differences between patients with or without microvascular complications for any of the MV parameters. ConclusionType 1 diabetes is a prothrombotic and proinflammatory disease state that, regardless of the presence of clinical microangiopathy, is associated with elevated levels of plasma MVs, in particular those of an endothelial origin. We have for the first time demonstrated that patients with type 1 diabetes have higher levels of HMGB1+ MVs. HMGB1 is an alarmin with potent proinflammatory effects that drive endothelial dysfunction, and it would therefore be of interest to further study the role of HMGB1+ MVs in the development of macrovascular complications in type 1 diabetes.

Increased release of microvesicles containing mitochondria is associated with the myeloid differentiation of AML-M5 leukaemia cells

Extensive literature has demonstrated that acute myeloid leukaemia (AML) cells show enhanced mitochondrial biogenesis and increased reliance on oxidative phosphorylation (OXPHOS) compared with normal hematopoietic progenitors, and one hallmark of AML leukaemia blasts is myeloid differentiation blockade. However, relatively few reports have linked these processes. Recent studies have indicated that therapies that overcome differentiation arrest represent an effective treatment strategy. Here, we identified that the disruption of the mitochondrial mass and energy metabolism promotes leukaemia cellular myeloid differentiation. In this study, we showed that acute monocytic leukaemia (AML-M5) cells package mitochondria in microvesicles (MVs) when MVs shed from membranes. Additionally, during myeloid differentiation, we report for the first time that differentiated leukaemia cells release more MVs than undifferentiated leukaemia cells. Targeting the formation of MVs using a specific inhibitor (Y-27632) restrained myeloid differentiation, suggesting that the increased release level of MVs plays an important role in regulating myeloid differentiation. Furthermore, the intracellular mitochondria and ATP levels were decreased after leukaemia cells overcame the differentiation blockade. Moreover, rotenone, which is used to inhibit the respiratory chain and ATP production, had a strong effect on myeloid differentiation in monocytic leukaemia cells. Collectively, these studies uncovered the relationship between mitochondrial function and myeloid differentiation and may provide more insight into the diagnosis and treatment of AML.

The microvesicle/CD36 complex triggers a prothrombotic phenotype in patients with non-valvular atrial fibrillation.

The mechanisms responsible for platelet activation, the prothrombotic state, in non‐valvular atrial fibrillation (NVAF) are still obscure. Microvesicles (MVs) can transfer various messages to target cells and may be helpful for exploring the detailed mechanisms. We aimed to investigate the possible mechanisms by which proatherogenic factors of NVAF contribute to platelet activation. Two hundred and ten patients with NVAF were stratified as being at ‘low to moderate risk’ or ‘high risk’ for stroke according to the CHADS2 score. Levels of platelet‐derived MVs (PMVs) and platelet activation were examined. CD36‐positive or CD36‐deficient human platelets were stimulated by MVs isolated from NVAF patients with or without various inhibitors in vitro. Levels of PMVs and platelet activation markers enhanced significantly in high‐risk patients. The MVs isolated from plasma of NVAF patients bound to platelet CD36 and activated platelets by phosphorylating the mitogen‐activated protein kinase 4/Jun N‐terminal kinase 2 (MKK4/JNK2) pathways. However, CD36 deficiency protected against MV‐induced activation of platelets. We reveal a possible mechanism of platelet activation in NVAF and suggest that the platelet CD36 might be an effective target in preventing the prothrombotic state in NVAF.

Plasma signature of apoptotic microvesicles is associated with endothelial dysfunction and plaque rupture in acute coronary syndromes

ObjectiveCirculating microvesicles (MV) are surrogate biomarkers of atherosclerosis. However, their role in acute coronary syndromes (ACS) has not been fully elucidated yet. We sought to examine the association of circulating apoptotic MVs with ACS pathophysiology. Approach and resultsOne hundred and fifty-three patients (n = 153) were included in the study; 49 patients with ST-elevation myocardial infarction (STEMI), 35 with non-STEMI (NSTEMI), 38 with unstable angina, 15 with stable coronary artery disease and 16 control individuals. Flow cytometry analysis was used to quantify circulating apoptotic/non-apoptotic (phospatidyloserine+/phospatidyloserine−) endothelial cell (EMV), red blood cell (RMV) and platelet (PMV) derived MV. Flow-mediated dilatation (FMD) of the brachial artery was assessed by ultrasound to estimate endothelial function. The inflammatory profile was assessed by serum C-reactive protein (hsCRP) levels. Apoptotic only (but not non-apoptotic) MV were increased in patients with ACS (EMV, P = 2.32 × 10−9; RMV, P = .0019; PMV, P = .01). Hierarchical clustering of the total population of ACS patients (n = 122) by circulating levels of phospatidyloserine+ EMV, RMV and PMV identified two discreet clusters of patients without any differences in traditional risk factors, but significant differences in brachial FMD (5.2% (2.5) vs. 4.1% (2.3), P < .05) that remained significant after adjustment for co-variates. The prevalence of STEMI, a surrogate for plaque rupture and vessel thrombotic occlusion, was significantly higher in the patient cluster with impaired endothelial function (60% vs 32%, P = .016, adjusted odds ratio for STEMI, 3.041, 95%CI, 1.160 to 7.968, p = .024). ConclusionOur findings indicate that the circulating levels of apoptotic MV are increased in ACS patients and their plasma profiles associate with endothelial dysfunction and thrombotic complications in ACS patients.

Sodium Intake, Circulating Microvesicles and Cardiovascular Outcomes in Type 2 Diabetes.

There is ongoing debate surrounding the complex relationship between dietary sodium intake and cardiovascular morbidity and mortality. The existing literature consists largely of observational studies that have demonstrated positive, negative, U-/J-shaped or unclear associations between sodium intake and cardiovascular outcomes. Our group and others have previously demonstrated an inverse relationship between dietary sodium intake and cardiovascular outcomes in people with type 2 diabetes. Increased activity of the renin-angiotensin-aldosterone system and sympathetic nervous system is postulated to contribute to these paradoxical findings through endothelial dysfunction, a precursor to the development of cardiovascular disease. Microvesicles are submicron (0.1 - 1.0μm) vesicles that form during cellular activation, injury or death with endothelial microvesicles being recognized markers of endothelial dysfunction. They are pathologically elevated in a variety of vascular-related conditions including type 2 diabetes. Lower habitual sodium intake in type 2 diabetes has been associated with higher pro-coagulant platelet microvesicles levels but not with endothelial microvesicles. Research utilizing endothelial microvesicles to evaluate the mechanistic relationship between dietary sodium intake and adverse cardiovascular outcomes in type 2 diabetes remains scarce.

Impact of left atrial appendage closure on circulating microvesicles levels: The MICROPLUG study

BackgroundLeft atrial appendage occlusion (LAAO) has emerged as a valid alternative to oral anticoagulation therapy for the prevention of systemic embolism in patients with non-valvular atrial fibrillation (NVAF). Microvesicles (MVs) are shed-membrane particles generated during various cellular types activation/apoptosis that carry out diverse biological effects. LAA has been suspected to be a potential source of MVs during AF, but the effects its occlusion on circulating MVs levels are unknown. MethodsN = 25 LAAO and n = 25 control patients who underwent coronary angiography were included. Blood samples were drawn before and 48 h after procedure for all. A third sample was collected 6 weeks after procedure in LAAO patients. In N = 10 extra patients, samples were collected from right atrium, LAA and pulmonary vein during LAAO procedure. Circulating AnnV + procoagulant, endothelial, platelets, red blood cells/RBC and leukocytes derived-MVs were measured using flow cytometry methods. ResultsIn the LAAO group, AnnV+, platelets, RBC, and leukocytes MVs were significantly increased following intervention, whereas only AnnV + MVs levels significantly rose in controls. The 6-w analysis showed that RBC-MVs and AnnV + MVs levels were still significantly elevated compared to baseline values in LAAO patients. The in-site analysis revealed that leukocytes and CD62e + endothelial-MVs were significantly higher in left atrial appendage compared to pulmonary vein, suggesting a local increased production.No major adverse event was observed in any patient post procedural course. ConclusionsLAAO impacts circulating MVs and might create mild pro-coagulant status and potential erythrocytes activation due to the device healing during the first weeks following intervention.

Shiga toxin signals via ATP and its effect is blocked by purinergic receptor antagonism

Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.

P3725Impact of left atrial appendage closure on circulating microvesicles levels: the MICROPLUG study

Abstract Background Percutaneous left atrial appendage occlusion (LAAO) has emerged as a valid alternative to oral anticoagulation therapy for the prevention of systemic embolism in patients with non-valvular atrial fibrillation (AF). Microvesicles (MVs) are shed-membrane particles generated during various cellular types activation/ apoptosis that carry out diverse biological effects, including procoagulant effects. Left atrial appendage has been suspected to be a potential source of MVs during AF, but the effects of LAAO on MVs production and circulating levels are unknown.The aim of this work study was to assess the variations of circulating MVs levels following LAAO. Methods The study includedn=25 LAAO patients and n=25 control patients who underwent coronary angiography. LAAO and control patients were treated by clopidogrel+ aspirin loading doses before procedures. Blood samples were drawn before antiplatelets therapy &amp; 2 days after for all. A third sample was collected 6 weeks after procedure in LAAO patients. In N=10 extra patients, blood samples were collected from right atrium, left appendage and pulmonary vein during LAAO procedure. Circulating procoagulant (AnnV+), endothelial (CD62e+), platelets (CD41+), red blood cells/RBC (CD235+), leukocytes (CD11+) derived-MVs were measured using flow cytometry methods. Results Control and LAAO groups baseline characteristics were comparable, except for the higher age &amp; incidence of previous stroke and lower incidence of coronary artery disease in LAAO patients. Baseline levels of the different microvesicles were comparable in both groups. In the LAAO group, we observed a significant increase of AnnV+ MVs (4355 [1712–8478] vs. 1798 [1006–2759] ev/μL, p=0.001), platelets (1615 [833–4772] vs. 802 [358–1376] ev/, p=0.005), RBC (207 [85–708] vs. 35 [5–84] ev/μL, p&lt;0.001), and leukocytes MVs (1368 [783–2319] vs. 1067 [827–1564] ev/μL, p=0.02) following intervention, whereas only AnnV+ MVs levels significantly rose in controls (3701 [2043–7017] vs. 1506 [1033–4899] ev/μL, p=0.03). The 6-w analysis showed that RBC-MVs (55 [8–182 ev/μL]and AnnV+ MVs levels (2468 [1813–5576 ev/μL]were still significantly increased compared to baseline values in LAAO patients (p&lt;0.05). The in-site analysis revealed that leukocyte MVs and CD62e+ endothelial-MVs were significantly higher in left atrial appendage compared to pulmonary vein (respectively 430 [26–700 vs. 161 [0–426] and 344 [22–723] vs. 200 [120–326] ev/μL, p&lt;0.05), suggesting a local increased production. No major adverse ischemic or bleeding event was observed in any patient post procedural course. Conclusions LAAO impact circulating MVs and could create mild pro-coagulant status, inflammation and potential erythrocytes activation due to device presence during the first 6 weeks following intervention. These results suggest that careful attention should be paid in the anti-platelet/anti-coagulant therapy in the post procedural course. Acknowledgement/Funding This work was funded by a research grant from the French Society of Cardiology and a research grant from St Jude/Abbott

P2253Intake of the omega 3 PUFAs formulation EPA:DHA 6:1 by aged rats reduced shedding of microvesicles from spleen-derived cultured leukocytes and their ability to promote senescence in endothelial cells

Abstract Introduction Ageing is associated with the appearance of endothelial senescence promoting endothelial dysfunction and, ultimately, cardiovascular events. Circulating microvesicles (MVs) of patients with acute coronary syndrome promoted premature endothelial senescence by stimulating the local angiotensin system. Omega 3 PUFAs have been shown to reduce the risk of cardiovascular disease in patients at high risk. Purpose This study investigated whether a 7-day intake of the omega 3 formulation EPA:DHA 6:1 by rats affects the level of MVs released by spleen-derived cultured leukocytes as well as their ability to promote premature senescence in target endothelial cells (ECs), and, if so, to clarify the underlying mechanism. Methods Middle-aged male Wistar rats (M, 48-week old) received 500 mg/kg/d of either EPA:DHA 6:1, EPA:DHA 1:1, or vehicle (CTL) for 7 days. Thereafter, spleen-derived leukocytes, a rich source of MVs, were prepared and cultured for 24 h. Cultured ECs were prepared from porcine coronary arteries. Senescence-associated β-galactosidase activity (SA-β-gal) was assessed by C12FDG, protein expression level by Western blot analysis, oxidative stress by dihydroethidium using confocal microscopy, and procoagulant MVs by prothrombinase assay. Spleen-derived leukocytes from untreated young (Y, 12-week) and old (O, 72-week) rats were also studied. Results Shedding of MVs by spleen-derived leukocytes significantly increased with increasing age. Incubation of ECs with leukocyte-derived MVs (10 nM Phtd Ser eq.) from M and O but not those from Y induced premature senescence after 48 h. The stimulatory effect of M-MVs was prevented by losartan and associated with oxidative stress. M-MVs induced an upregulation of senescence markers (p16, p21, p53), pro-atherothrombotic markers (VCAM-1, ICAM-1, tissue factor), the pro-inflammatory marker cyclooxygenase-2 (COX-2) but not COX-1, and of the angiotensin system (angiotensin-converting enzyme and type 1 angiotensin receptor), whereas endothelial NO synthase was down-regulated. A one-week intake of EPA:DHA 1:1 and 6:1 by M rats decreased the leukocyte-derived MVs shedding by about 14% and 24%, and EPA:DHA 6:1 reduced their ability to induce ECs senescence by 38%. The stimulatory effect of M-MVs on the expression of target proteins was also observed with those from the EPA:DHA 1:1 but not with those from the 6:1 group. Conclusion These findings indicate that ingestion of EPA:DHA 6:1 by middle-aged rats reduces not only the shedding of MVs by spleen-derived leukocytes but also their ability to induce pro-senescent, pro-thrombotic and pro-inflammatory responses in endothelial cells most likely by decreasing the local angiotensin system. They further suggest that EPA:DHA 6:1 may help to delay ageing-related endothelial dysfunction. Acknowledgement/Funding Unrestricted research grant from PIVOTAL Therapeutics Inc.

Changes in plasma circulating microvesicles in patients with HCV-related cirrhosis after treatment with direct-acting antivirals.

The eradication of Hepatitis C (HCV) infection by direct-acting antiviral (DAAs) agents has been linked to an amelioration of liver synthesis and regression of fibrosis. Although changes in number and type of circulating microvesicles (MVs) have been reported in cirrhosis, conclusive data on the effect of DAAs treatment on MVs profile in HCV cirrhotic patients remain scarce. We measured the levels of endothelial, platelet and hepatocyte MVs, as well as MVs-expressing versican core protein (VCAN+) in patients with HCV-related cirrhosis at baseline, end of treatment (EOT), at 12, 24 and 48weeks (W) after EOT by new generation flow cytometry. Fifty-eight patients were enrolled (86% Child's A). MVs were increased at EOT vs baseline, though only platelet MVs revealed a statistically significant difference (P<.01). MV levels did not change significantly after EOT notwithstanding a steady downward trend towards baseline levels. Conversely, VCAN+MVs dropped significantly at EOT (P<.001) and remained low throughout the follow-up. Hepatocyte MVs significantly correlated with liver stiffness (r=.40, P=.0021). Eight composite outcomes occurred during the 1-year follow-up: three portal vein thromboses (PVTs), two hepatocellular carcinomas (HCCs) and three liver decompensation. Child's B, the presence of F2 oesophageal varices (OR for interaction 19.2 [95% CI 1.45-253.7], P=.023) and platelet MVs (OR 1.026 [95% CI 1.00-1.05, P=.023) correlated significantly with clinical outcomes. VCAN+MVs appear to mirror the profibrotic status of the cirrhotic disease; hepatocyte MVs correlate with liver stiffness and increased platelet MV levels could be associated with a worse clinical outcome.

Increased erythrocyte- and platelet-derived microvesicles in newly diagnosed type 2 diabetes mellitus.

To investigate the thrombotic microenvironment in early stages of type 2 diabetes mellitus measuring platelet-derived, endothelial-derived and erythrocyte-derived microvesicles. We recruited 50 newly diagnosed type 2 diabetes mellitus patients who did not receive glucose-lowering treatment except for metformin and 25 matched non-type 2 diabetes mellitus volunteers. Microvesicles were measured with flow cytometry, glycated haemoglobin with high-performance liquid chromatography and advanced glycation end products with enzyme-linked immunosorbent assay. Type 2 diabetes mellitus patients showed significantly higher levels of platelet-derived microvesicles [195/μL (115-409) vs 110/μL (73-150), p = 0.001] and erythrocyte-derived microvesicles [26/μL (9-100) vs 9/μL (4-25), p = 0.007] compared to non-type 2 diabetes mellitus individuals. Platelet-derived microvesicles were positively associated with fasting blood glucose (p = 0.026) and glycated haemoglobin (p = 0.002). Erythrocyte-derived microvesicles were also positively associated with fasting blood glucose (p = 0.018) but not with glycated haemoglobin (p = 0.193). No significant association was observed between platelet-derived microvesicles (p = 0.126) or erythrocyte-derived microvesicles (p = 0.857) and advanced glycation end products. Erythrocyte-derived microvesicles predicted the presence of type 2 diabetes mellitus, independently of platelet-derived microvesicles. In newly diagnosed type 2 diabetes mellitus, ongoing atherothrombosis is evident during the early stages as evidenced by increased microvesicles levels. Furthermore, the association with glycemic profile suggests that microvesicles represent not only a novel mechanism by which hyperglycemia amplifies thrombotic tendency in type 2 diabetes mellitus but also early markers of thrombosis highlighting the need for earlier management of hyperglycemia.

Circulating microvesicles in snakebite patients with microangiopathy

BackgroundVenom‐induced consumption coagulopathy is a common consequence of snake envenoming that can lead to life‐threatening hemorrhage, and is associated with microangiopathic hemolytic anemia (MAHA), acute kidney injury and thrombocytopenia. The role of microvesicles (MV) in snakebite patients has not been previously investigated. ObjectiveTo compare changes in subsets of circulating MV levels in snakebite patients with venom induced consumption coagulopathy and with or without microangiopathic hemolysis to those of healthy controls. MethodsThis study used samples from patients recruited to the Australian Snakebite Project (ASP) with snake envenoming, including bites by brown snakes, tiger snakes, and taipans. Citrated blood from envenomed patients was collected, processed, and stored according to a national standardized protocol. Full blood count and coagulation parameters were measured as per routine clinical care and blood films were examined for evidence of hemolysis. Baseline coagulation parameters were measured on a Behring Coagulation System. Flow cytometry was performed to detect CD41a (platelet), CD62e (endothelial), and glycophorin (red cell) MV. The results were analyzed using BD software and appropriate statistical tools. Results and ConclusionsThe red cell MV in snakebite patients with MAHA (n = 13) were significantly higher than those without MAHA (n = 17) while there was no significant difference in platelet MV levels between the snakebite patients with and without MAHA. Interestingly, the endothelial MV were reduced in all snakebite patient samples compared to the control samples. Measuring red cell MV at presentation could be useful as a predictive marker for MAHA in patients with snakebites.

A Novel Plasma Panel for the Assessment of Chronic Liver Disease Severity

Background: Hepatocytes produce a variety of clotting and fibrinolytic factors. In chronic liver disease (CLD), biosynthesis of these factors is often altered leading to hemostatic aberrations. Recently, the usefulness of PT/INR - one of the five parameters used in the Child-Pugh (CP) scoring system - has been questioned. In this retrospective study, we explored the patterns of plasma alterations relative to CP scores to establish a plasma-based panel that can help evaluate the extent of CLD severity without the use of PT/INR and/or such parameters as the extent of ascites and hepatic encephalopathy.Methods: Blood was collected via venipuncture (0.129 mol/l sodium citrate) from CLD patients suffering from cirrhosis due to untreated HCV and/or excessive alcohol consumption, that were previously assigned a CP score: CP-A, n=12; CP-B, n=19; CP-C, n=11. Healthy control subjects (n=30) were age and sex matched; all subjects gave informed consent. Within 2 h post-collection, specimens were centrifuged for 15 min at 1500 x g, and then 13000 x g for 2 min to obtain plasma that was immediately frozen at -80°C until used. The following was measured: pro-coagulant phospholipid, fXa, D-dimer, fVIII, sP-selectin, thrombomodulin, protein C, protein S, alternatively spliced Tissue Factor (asTF), free TFPI, TF/TFPI ratio, and TFPI/protein S ratio. Thrombin generation tests (TGT) with and without thrombomodulin were performed. To interrogate the biology of CLD, patient plasma was also analyzed for the levels of microvesicles (MVs) of various origin: endothelium (CD31+/CD41-), monocytes (CD14+), leukocytes (CD11b+), neutrophils (CD16+), platelets (CD41a+, CD41a+/CD62P+, CD62P+), and exosomes (CD63+). Platelet counts and the levels of Annexin V+ MVs were also evaluated. Results were analyzed using GraphPad Prism v.6.03, and the parameters that were significantly different between control and CLD plasma were further evaluated. A system was developed with specified cutoffs for each parameter, derived using clinically normal ranges as well as the data generated in this study, to compile a composite score for CLD severity.Results: CLD plasma had endogenous thrombin potential comparable to that of control plasma; however, the rate of thrombin generation and peak thrombin generation diminished with increased CLD severity (both p < 0.0001), prolonging the time until peak (p < 0.01) as assessed by TGT. Of the 14 remaining parameters investigated in CLD and control plasma, the levels of fVIII, D-dimer, and asTF increased with CLD severity while protein C, fV, protein S, and sP-selectin levels decreased (Table 1) and in combination, these 7 parameters were able to predict the previously assigned CP scores with high accuracy. Our scoring system assigns a numeric value (0, 1, or 2) for each parameter; a cumulative score of <2 is normal, 2-3 CP-A, 4-7 CP-B, and ≥8 CP-C (Table 2). The system was 97.2% accurate at predicting the CP score: of the 72 plasma samples analyzed, there was only one false positive (a control scored as a CP-A), and one CP-C sample scored as CP-B. Omission of any of the 7 components markedly lowered the system's accuracy. Levels of CD31+/CD41-, CD62+, CD63+, and CD16+ MVs rose with disease severity (p < 0.0001 for all) while CD41a+ MVs decreased (p < 0.0001). Of the panel's 7 components, asTF is relatively novel and so asTF's association with various markers was further examined. There was a positive correlation between asTF and D-dimer (r = 0.305, p = 0.05) and asTF and CD63 (r = 0.346, p = 0.025); asTF negatively correlated with CD14+ MVs (r = -0.348, p = 0.024). There was a strong and highly significant positive correlation between asTF and CD31+/CD41- MVs (r = 0.630, p = 7.81x10-6).Conclusions: Here we report a novel plasma panel with potential utility in assessing CLD severity in a point-of-care setting. Our scoring system exhibits 97.2% accuracy and does not require clinical assessment beyond plasma analysis. asTF levels positively correlate with those of CD31+/CD41- MVs and both increase with CLD severity; this is in agreement with earlier reports of inflammatory cytokines eliciting asTF secretion from endothelial cells. Larger-scale prospective studies are warranted to validate our findings. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Abstract 16662: Microvesicle Release During Exercise-Induced Cardiovascular Stress in Young Adult Hypertension

Introduction: Microvesicles are elevated in the circulation during cardiac stress; a response that is blunted in those with cardiovascular disease. Microvesicles play both activating and regulatory roles in the pathogenesis of hypertension, and their release is believed to be a protective mechanism during cardiac stress. Hypothesis: We hypothesised that microvesicle release during exercise-induced cardiac stress differs in young adults with and without hypertension. Methods: Microvesicle release was measured in 106 participants (79 normotensive, 27 hypertensive). Blood samples were obtained during maximal exercise testing at three time-points: before, immediately post and following 20 minutes recovery. The level of microvesicle procoagulant activity was determined in all participants. Microvesicles were phenotyped by flow cytometry in a subset of participants. Results: Exercise was associated with a significant elevation in the total procoagulant activity of microvesicles (p&lt;0.001) and this increase was significantly higher in the normotensive group than the hypertensives (median(IQR) +0.7(0.0-2.1) vs -0.1(-0.4-0.4), p=0.001). Circulating procoagulant, platelet, endothelial, leucocyte, granulocyte and monocyte-derived microvesicles were shown to be elevated during exercise (p&lt;0.05). The elevation in endothelial and leucocyte microvesicle levels returned to baseline within 20 minutes recovery. The significant elevation in procoagulant, platelet, granulocyte and monocyte-derived microvesicles was only seen in the normotensive participants, not in those with hypertension. Furthermore, amongst the normotensive participants, those with a blunted release of platelet microvesicles had higher peak diastolic blood pressures (86.8±8.4 vs 73.5±14.5, p=0.028) during exercise and increased global arterial stiffness measured at rest by the cardio-ankle vascular index (CAVI: 6.1±0.6 vs 5.4±0.5, p=0.022). Conclusions: In young adult hypertension there is a blunted release of microvesicles during exercise-induced cardiac stress. Normotensive young adults with a blunted platelet microvesicle release demonstrate early markers of future hypertension risk with greater peripheral resistance and arterial stiffness.

Phosphatidylserine expressing microvesicles in relation to microvascular complications in type 1 diabetes

IntroductionType 1 diabetes is a prothrombotic state strongly linked to vascular complications. The role of microvesicles (MVs) as mediators and potential biomarkers in microangiopathy in type 1 diabetes remains unclear. Materials and methodsMV levels in plasma samples from 106 patients with type 1 diabetes with microangiopathy, 130 patients without microangiopathy and 100 healthy controls were analysed using flow cytometry. Phosphatidylserine (PS) expression in MVs was assessed by lactadherin, and the ability of MVs to induce thrombin generation was investigated in vitro. Endogenous plasma lactadherin levels were measured using ELISA. ResultsPatients with type 1 diabetes had higher MV levels compared to healthy controls, with no significant differences between patients with and without microangiopathy. MV-induced thrombin generation in normal-pooled plasma was blocked by addition of lactadherin. Endogenous lactadherin levels were higher in patients compared to controls, and the highest levels were found in patients with microangiopathy. Plasma lactadherin levels did not correlate with levels of PS positive/negative MVs. ConclusionPatients with type 1 diabetes with and without microangiopathy have higher levels of circulating MVs than healthy controls, probably reflecting higher cellular activation and turnover. However, we found no associations between clinical microangiopathy and levels of MVs in total or PS-expressing MVs. Plasma levels of lactadherin, which is a glycoprotein important in the clearance of cells and MVs, are increased in patients with type 1 diabetes and correlate with microangiopathy.

Bariatric surgery reduces CD36-bearing microvesicles of endothelial and monocyte origin

BackgroundBariatric surgery is a widely adopted treatment for obesity and its secondary complications. In the past decade, microvesicles (MVs) and CD36 have increasingly been considered as possible biomarkers for obesity, the metabolic syndrome (MetSy), type 2 diabetes mellitus (T2DM). Thus, the purpose of this study was to investigate how weight loss resulting from bariatric surgery affects levels of specific MV phenotypes and their expression of CD36 scavenger receptor. Additionally, we hypothesised that subjects with MetSy had higher baseline concentrations of investigated MV phenotypes.MethodsTwenty individuals undergoing Roux-en-Y gastric bypass surgery were evaluated before and 3 months after surgery. MVs were characterised by flow cytometry at both time points and defined as lactadherin-binding particles within a 100-1000 nm size gate. MVs of monocyte (CD14) and endothelial (CD62E) origin were defined by cell-specific markers, and their expression of CD36 was investigated.ResultsFollowing bariatric surgery, subjects incurred an average BMI reduction (delta) of − 8.4 ± 1.4 (p < 0.0001). Significant reductions were observed for the total MVs (− 66.55%, p = 0.0017) and MVs of monocyte (− 36.11%, p = 0.0056) and endothelial (− 40.10%, p = 0.0007) origins. Although the bulk of CD36-bearing MVs were unaltered, significant reductions were observed for CD36-bearing MVs of monocyte (− 60.04%, p = 0.0192) and endothelial (− 54.93%, p = 0.04) origin. No differences in levels of MVs were identified between subjects who presented with MetSy at baseline (n = 13) and those that did not (n = 7).ConclusionBariatric surgery resulted in significantly altered levels of CD36-bearing MVs of monocyte and endothelial origin. This likely reflects improvements in ectopic fat distribution, plasma lipid profile, low-grade inflammation, and oxidative stress following weight loss. Conversely, however, the presence of MetSy at baseline had no impact on MV phenotypes.

MicroRNA-1 and Circulating Microvesicles Mediate the Protective Effects of Dantonic in Acute Myocardial Infarction Rat Models.

Aim: To investigate the protective effect of dantonic in ischemic myocardial damage by evaluating the expression of circulating microvesicles (MVs) and microRNA-1 (miR-1) in two animal models.Methods: Two animal models of myocardial ischemia were established that were isoproterenol-induced myocardial ischemia (ISO-AMI) rat model and the acute myocardial infarction rat model induced by ligation of the left anterior descending coronary artery (LAD-AMI) of rat. To investigate the protective effect of dantonic, we observed the myocardial infarction size, creatine kinase (CK), lactate dehydrogenase (LDH), aspartate aminotransferase (AST) activities, cardiac troponin I (cTnI) level in serum, and the plasma levels of miR-1 and MVs.Results: The results showed that pretreatment with dantonic significantly attenuated the LAD-AMI induced myocardial damage by decreasing the size of myocardial infarction, CK, LDH, AST activities, and cTnI level in serum. High dose dantonic treatment could significantly abrogate the increased plasma levels of miR-1 and MVs as compared to the LAD rat model. In addition, pretreatment with dantonic also showed a significant myocardial protective effect through reducing the expression levels of CK, LDH, and AST as compared to the ISO-AMI model. Whereas the cTnI level was no significant difference between model group and control group, suggesting that the model caused less myocardial damage. In the ISO-induced myocardial ischemia model, there is no significant difference between the model group with the control group of MVs and miR-1 levels. This may be that miR-1 is reported as a biomarker of acute myocardial infarction. The pathological changes of IOS-induced acute myocardial ischemia model are also different from those of acute myocardial infarction.Conclusion: Dantonic showed the protective effect in these two ischemic myocardial injury rat models, whereas the circulating miR-1 and MVs levels were only ameliorated in the LAD rat model.

Hepatocyte microvesicle levels improve prediction of mortality in patients with cirrhosis.

Circulating MV levels cannot identify patients with HVPG ≥10 mm Hg; by contrast, hepatocyte MV levels strongly improve prediction of 6-month mortality in patients with advanced chronic liver disease; therapies associated with decreased levels of circulating hepatocyte MV might be attractive strategies in patients with severe cirrhosis. (Hepatology 2018).

Postprandial Increase in Blood Plasma Levels of Tissue Factor-Bearing (and Other) Microvesicles Measured by Flow Cytometry: Fact or Artifact?

Tissue factor (TF)–bearing microvesicles (MVs) and exosomes may play a role in hemostasis and thrombosis. MVs may be quantified by flow cytometry (FC)–based detection of phosphatidylserine (PS)-positive submicron particles carrying specific antigens, although interference from lipoproteins complicates this approach. In this study, we evaluated the effect of food intake on blood levels of TF-bearing particles measured by FC and small extracellular vesicles (EVs) measured by a protein microarray–based test termed EV Array. Platelet-free plasma (PFP) was obtained from 20 healthy persons in the fasting state and 75 minutes after consumption of a meal. Postprandial changes in the concentration of PS-positive particles, including subgroups binding labeled antibodies against TF, CD41, CD146, and CD62E, respectively (FC), small EVs (EV Array), and TF antigen and procoagulant phospholipids (PPLs) were measured. Furthermore, we tested the effect on FC results of in vitro addition of lipoproteins to fasting PFP. We found significantly increased plasma concentrations of PS-positive particles and all examined subgroups postprandially, while no changes in small EVs, PPL, or TF antigen levels were found. Levels of all types of particles measured by FC were also elevated by lipoprotein spiking. In conclusion, meal consumption as well as in vitro addition of lipoproteins to fasting plasma induces increased levels of PS-positive particles as measured by FC, including TF-positive subtypes and subtypes exposing other antigens. While the observed postprandial increase may to some extent reflect elevated MV levels, our results indicate a substantial interference from lipoproteins.