Plasma Microparticles Research Articles

Plasma microparticles from patients with systemic lupus erythematosus modulate the content of miRNAs in U937 cells.

In systemic lupus erythematosus (SLE), the clearance of apoptotic cells and microparticles (MPs) is reduced. Some MPs contain molecules that can modulate immune responses. This study aimed to evaluate the presence of miR-126 and miR-146a in plasma MPs of patients with SLE (SLE MPs) and analyse the ability of MPs to modulate some events in the promonocytic U937 cell line. Circulating MPs were isolated from plasma samples of healthy controls (HCs), patients with SLE and other autoimmune diseases (OAD). MPs were analysed for size and cell origin by flow cytometry and content of miR-126 and miR-146a by RT-qPCR. MPs were then added to U937 cell cultures to evaluate changes in cell phenotype, cytokine expression, content of miR-126 and miR-146a, and levels of IRF5. Patients with active SLE (aSLE) showed an increase in concentration of plasma MPs that positively correlated with the SLEDAI (SLE Disease Activity Index) score. CD14+ MPs were significantly more abundant in patients with SLE than HCs. SLE MPs contained decreased levels of miR-146a, but the miR-126 content in aSLE MPs was increased. The miR-126 content in SLE MPs correlated positively with the SLEDAI score. The treatment of U937 cells with MPs from HCs and patients induced reduced expression of HLA-DR, CD18 and CD119, increased frequency of IL-6+ and TNF-α+ cells, accumulation of IL-8 in culture supernatants, increased miR-126levels and decreased miR-146a content, but no change in the expression of IRF5. These findings suggest that plasma MPs, especially SLE MPs, could modulate some biological events in U937 cells.

Abstract P031: Endothelial Microparticles In Pediatric Obesity

Introduction: Endothelial activation represents one of the earliest signs of the atherosclerotic process. Microparticles (MPs) in plasma, particularly endothelial MPs (EMPs) are reproducible biomarkers of endothelial dysfunction with a simple blood draw. The goal of this study was to examine the association of obesity with MPs, EMPs (CD31+/CD41– and/or CD 144+/CD31–), and activated EMPs (CD106+). Hypothesis: We hypothesized that higher body mass index (BMI), body fat percent (BF%), and visceral adipose tissue (VAT) would be associated with higher levels of MPs, EMPs, and activated EMPs. Methods: This was a cross-sectional study of 280 children and adolescents ages 8-18 years old. Whole venous blood was collected and processed for platelet free-plasma (PFP) collection less than 3 hours after being drawn. PFP samples were prepared using serial centrifugations and then frozen as 100 μl aliquots at -80 ° C. MPs were measured using flow cytometry with size ranging from 0.3 to 1.0 μm. EMPs were defined as MP with CD31+/CD41– and/or CD 144+/CD31–. Active EMP was identified as EMP with CD 106+. BF% and VAT were measured using dual X-ray absorptiometry. Linear (total MPs and EMPs) and logistic (activated EMPs) regression models were used to examine the associations with adiposity group, BF%, and VAT, adjusting for Tanner stage, sex, and race. Results: The mean age of the cohort was 12.7 years, consisting of 147 females and BMI distribution: 112 normal-weight, 77 overweight/obesity, 91 severe obesity. Youth with overweight/obesity and severe obesity had higher numbers of total MPs (28,690 per μL PFP/g [-2,315, 59,695], p=0.07 and 52,744 per μL PFP/g [22,163, 83,324], p<0.001, respectively) and total EMPs (350 per μL PFP/g [81, 618], p=0.011 and 278 per μL PFP/g [13, 543], p=0.040, respectively) compared to peers with normal weight. In the entire cohort, higher BF% was associated with higher MPs (1,646 per μL PFP per percent [509, 2,783], p=0.005) and EMPs (11 per μL PFP per percent [2, 21], p=0.023), and higher VAT was associated with higher MPs (34 per μL PFP/g [11, 57], p=0.004) but not EMPs. Logistic regression analysis showed higher odds of active EMPs (odds ratio, 1.72 [1.0, 2.97], p=0.0502) in those with a BMI >85 th percentile compared to youth with a BMI <85 th percentile. In the entire cohort, BF% (odds ratio, 1.02 [1.00, 1.04], p=0.108) and VAT (odds ratio, 1.00 [1.00, 1.00], p=0.472) were not significantly associated with active EMPs. Conclusions: Higher levels of endothelial cell damage and activation were associated with obesity and excess adiposity in youth. The potential role of MPs and EMPs as useful biomarkers of cardiovascular disease risk merits further study.

Circulating microparticles in patients with chronic hepatitis C and changes during direct-acting antiviral therapy.

Microparticles (MPs) are heterogeneous vesicles derived from membranes of different cells. Between 70 to 90% of MPs detected in blood originate from platelets. The release of MPs is associated with proinflammatory and procoagulant states. Elevated levels of MPs have been found in different diseases. We investigated MPs levels in patients with chronic hepatitis C (CHC) and changes in level during treatment using direct-acting antivirotics (DAA). Thirty-six patients with CHC and forty healthy volunteers were included in the study. Concentrations of MPs were determined indirectly by measuring their procoagulant activity in plasma at baseline, end of therapy (EOT), and 12 weeks after EOT when the sustained virological response was assessed (SVR12). All patients achieved SVR12, which was associated with rapid improvement of markers of liver damage and function as well as liver stiffness (P=0.002). MPs levels were significantly higher in CHC patients than in healthy volunteers (P<0.001). No statistically significant decrease was found observed between baseline and SVR12 (P=0,330). Analysis of subpopulations with minimal fibrosis F0-1 (P=0.647), advanced fibrosis F2-4 (P=0.370), women(P=0.847), men (P=0.164) and genotype 1 (P=0.077) showed no significant changes during the follow-up period. MPs levels are higher in CHC patients and remain elevated shortly after achieving SVR. Higher concentrations of MPs in plasma are probably caused by a chronic uncontrolled exaggerated inflammatory response caused by CHC. Longer observation would probably confirm the significance of MPs levels decrease because normalization of liver function, inflammation, and structure after SVR requires more than 12 weeks.

Microparticles: a link to increased thrombin generation.

Circulating microparticles in human plasma may play a significant role in thrombogenesis because they carry the initiator of blood coagulation, tissue factor. Microparticles in blood are derived from diverse cell types, including erythrocytes, endothelial cells and platelets. Thrombin generation is an important part of the coagulation system and might be influenced by the presence of microparticles in the circulation. With this study, we determined the contribution of microparticles to increased thrombin generation in plasma samples received for thrombophilia workup and compare that with normal plasma. Microparticles were isolated from 50 plasma samples with increased thrombin generation and 20 plasma samples with normal thrombin generation, using filtration. Thrombin generation assay were performed by adding a low concentration of tissue factor-containing phospholipids and a fluorescence substrate for thrombin formation to plasma samples and measuring fluorescence at 1-min intervals over a period of 90 min on all samples (with and without the presence of microparticles). The peak thrombin, velocity-index and area under the curve were calculated. Microparticles contribute to the different parameters in samples with increased thrombin generation as follows: 50 ± 19% for peak thrombin, 58 ± 24% for velocity-index and 35 ± 13% for area under the curve. Microparticles did not contribute to thrombin generation in plasma samples with normal thrombin generation. Microparticles play a significant role in coagulation and contribute largely to increased thrombin generation in plasma; however, microparticles do not contribute to coagulation in the plasma of participants with normal thrombin generation.

Measurement of canine blood microparticles by flow cytometry: effect of anticoagulants and staining reagents.

Microparticles (MPs) are released from budding plasma membranes into body fluids. The use of flow cytometry for the measurement of MP in canines has not been standardized. In this fundamental study, we compared the effect of anticoagulant agents, such as acid-citrate-dextrose (ACD) and heparin on the measurement of canine MPs in platelet-free plasma (PFP) using flow cytometry. In addition, we used annexin V, carboxyfluorescein succinimidyl ester (CFSE), or calcein tetraacetoxymethyl ester (calcein-AM), and explored the characteristics of the staining reagents in MP detection using flow cytometry. We were able to measure canine MPs in PFP prepared from ACD-anticoagulated blood using flow cytometry, in which the highest positive rate for fluorescent staining was observed when CFSE was used.

Internalization of cell-derived microparticles triggers endothelial pro-inflammatory responses.

Increased numbers of circulating microparticles (MPs) have long been documented in thalassemia and are considered as a contributing factor in developing the thromboembolic events (TEEs), which are associated with endothelial dysfunction. Indeed, the cellular and molecular mechanisms by which MPs and endothelial cells interact and their consequences remain poorly investigated. The present study aims to compare the biological effects of MPs obtained from healthy subjects and β-thalassemia/HbE patients on endothelial pro-inflammatory responses. MPs isolated from plasma by two-step centrifugation from 10 healthy donors, 19 splenectomized and 30 non-splenectomized β-thalassemia/HbE patients were first characterized for their cellular origins, then counted and incubated with primary human umbilical vein endothelial cells (HUVECs). Internalization of MPs into HUVECs and their induction on endothelial cell activation and pro-inflammatory responses were determined. MPs either from healthy or β-thalassemia/HbE patients could become internalized into endothelial cells, but unlike MPs from healthy donors and non-splenectomized patients, MPs from splenectomized patients were the most active and induced the 2-fold up-regulation of pro-inflammatory genes, IL1B, CXCL8, and CCL2 and 4-fold increase in interleukin-1β. In addition, MPs from both healthy subjects and splenectomized patients at 106/ml failed to trigger the secretion of endothelial IL-6 and IL-8 while higher MP concentration at 5 × 10⁶/ml significantly induced this secretion. Plasma MPs isolated from splenectomized β-thalassemia/HbE patients are capable of triggering pro-inflammatory responses from endothelial cells reflected at both gene and protein levels.

Level of Microparticles in Patients with Essential Thrombocythemia and Its Relation with JAK2V617F Mutation

To detect the levels of microparticles (MP) in plasma of patients with esseutial thrombo-cythermia(ET) and analyze the relationship between the JAK2V617F mutant and MP in ET patients. The numerical values of MPs were analysed by using flow cytometry. Venous blood of 56 ET patients and 28 healthy persons was collected in the morning and anticoagulated with sodium citrate (1∶9). The RMP, PMP, TF+MP and EMP were detected by FCM using phycoerythrin (PE)-conjugated monoclonal antibodies to CD235a for red blood cells, CD61 for platelets, CD142 for tissue factor (TF) and CD62E for endothelial cells, respectively. Forward scatter was set in scale using fluorescent microspheres of 0.8 μm. Standard fluorescent microbeads (0-0.8 μm) in diameter were used to set the microparticles gate. Genomic DNA was extracted from mononuclear cells by using a commercial DNA isolation kit and amplified by allele specific polymerase chain reaction (PCR). According to the size of molecular weight, the amplified products were separated by electrophoresis on a 2% agarose gel to screen 26 JAK2V617F mutations. The detection results showed that the MP levels in ET group were higher than those in normal control group: RMP (157.2±304.9/μl vs 21.3±18.4/μl), PMP (1378.9±2454/μl vs 113.8±97.1/μl), TF+MP (123±354.6/μl vs 21±15.7/μl) and EMP (1434.7±2601.9/μl vs 291.7±297.7/μl) (P<0.05). In 8 patients with thrombus, the levels of these four kinds of MP were higher than those of controls without thrombus: RMP (258.2±327.7/μl vs 55.6±63.8/μl), PMP (1853±1874.7/μl vs 444.7±712/μl), TF+MP (120.4±112.5/μl vs 60.3±128.3/μl) and EMP (1637.1±1563.5/μl vs 629.4±1000.2/μl) (P<0.05). The levels of those 4 kinds of MP in 17 patients with splenomegaly were significantly higher than those in 11 patients without splenomegaly: RMP (306.8±491.1/μl vs 59.3±51/μl), PMP (2944.8±3767.6/μl vs 334.8±420.2/μl), TF+MP (162.9±166.8/μl vs 31.0±28.7/μl) and EMP (3031.8±4048.8/μl vs 701.5±729.2/μl) (P<0.05). The level of other MP showed no difference between MPN patients with JAK2V617F mutation and without JAK2V617F mutation (P>0.05), except TF+MP (154.7±516.3/μl vs 100.5±126.6/μl) (P<0.05). The numerical values of MP detected are more in ET patients than those in healthy controls. The number of MP is higher in patients with thrombus than that without thrombus, so do in patients with splenomegaly and without splenomegaly. Patients with JAK2V617F mutation show higher number of TF+MP than that without JAK2V617F mutation. But the other three kinds of MP show no this difference.

Systematic development of a bioanalytical UPLC-MS/MS method for estimation of risperidone and its active metabolite in long-acting microsphere formulation in rat plasma

A systematic approach to develop a UPLC-MS/MS method was applied for quantifying of risperidone (RISP), its active metabolite, 9-hydroxy risperidone (9-OH-RISP) and internal standard (propranolol) in rat plasma. Liquid-liquid extraction was performed using methyl tert-butyl ether for quantification of drug and its active metabolite by MS detection in the positive ion mode. Acquity UPLC system with BEH C18 (2.1 mm × 100 mm, particle size 1.7 μm) column was used along with acetonitrile (0.1% formic acid)–2 mM (milli mole) ammonium acetate in isocratic condition was used as the mobile phase. Detection was performed by multiple reactions monitoring with precursor-to-product ion transitions with m/z 411.2 → 191.0 for RISP, m/z 427.2 → 207.0 for 9-OH-RISP and m/z 260.1 → 116.0 for IS. The method was validated as per the FDA guidance on bioanalytical method validation. Linearity (r2 = 0.999) was observed in the drug concentration ranging between 0.1 and 50 ng mL−1, while all other parameters were found to be within the acceptable ranges. Method robustness was optimized by Box-Behnken design to monitor the influential variables to achieve maximal recovery of the analytes in the rat plasma. Pharmacokinetic evaluation of the analytes from long-acting microparticles in rat plasma showed two peaks indicating an initial burst effect within 24 h of administration followed by controlled drug release pattern upto 45 days, while marketed formulation (Risperdal Consta®) showed no plasma concentration during the lag-time of 21 days followed by maximal drug absorption between 28 and 40 days.

Platelet activation in rabbits with decompression sickness

Platelets are the most easily altered type of peripheral blood cell in decompression sickness (DCS), which can feature decreased platelet count and the appearance of platelet microparticles in plasma. We hypothesized that DCS results in platelet activation in the bloodstream. The present study was carried out on 45 rabbits. The platelet count and concentration of plasma platelet markers were determined in 35 rabbits; the platelet shape was observed under scanning electron microscope in 10 rabbits. All indexes were collected at two points: 24 hours before the simulated dive and 30 minutes after the simulated dive. Platelet count decreased noticeably after DCS, from 380.10 ± 73.61 (G/L) to 330.23 ± 115.72 (G/L), a change of approximately -13.49 ± 25.57 (%). Platelet count was further decreased in the severe DCS group (a change of -45.99 ± 18.57%). Platelet count after DCS was proportional to the survival time of the rabbits after DCS. The concentration of two plasma platelet markers (PF4 and BTG) did not demonstrate statistically significant change at 30 minutes after DCS. However, platelet shape was changed, and the following features were observed: oblong, distortion, flattening shape, sticking together, mixing of membrane, and abundance of pseudopods with a 100- to 200-nm diameter. We conclude there is platelet activation in the bloodstream in cases of DCS.

Prognostic significance of tissue factor in patients with pancreatic cancer: a systematic review protocol.

IntroductionPancreatic cancer is a highly aggressive digestive system tumour with poor prognosis. Venous thromboembolism (VTE) is a well-known complication of pancreatic cancer, and tissue factor (TF) contributes to the generation of a hypercoagulable state and thrombotic disease in pancreatic cancer. Several studies showed that an elevated TF level was related to the development of VTE and influenced the survival of patients with pancreatic cancer. Thus, we wish to conduct a systematic review of literature to clarify the prognostic significance of TF in pancreatic cancer.Methods and analysisStudies comparing the circulating microparticle-associated TF (MP TF) level between patients who had pancreatic cancer with and without VTE will be included to evaluate the roles of TF in VTE development. Studies comparing the survival data between patients with high TF expression and low TF expression will also be included to explore the association of TF expression with patient survival. The outcomes are plasma MP TF level and survival endpoints (overall and progression-free survival), respectively. Primary studies of any type published in English will be included. Two reviewers will search Medline, EMBASE and Cochrane databases from inception to June 2020, retrieve relevant studies, and independently select the literatures and extract data from the included studies. The quality of each included study will be assessed by the Newcastle-Ottawa Scale score. The HR and 95% CI of each study will be pooled for survival outcome, and the standardised mean difference (SMD) with 95% CIs will be used for continuous outcomes. If meta-analysis is inappropriate, the result will only be reported qualitatively. Subgroup and sensitivity analyses will be considered to identify sources of heterogeneity. The Grades of Recommendation, Assessment, Development and Evaluation method will be applied to assess the level of evidence of this systematic review.Ethics and disseminationThere are no concerning ethical issues. The results will be published.PROSPERO registration numberCRD42019133665.

Experimental study of the nonreciprocal effective interactions between microparticles in an anisotropic plasma

There is a variety of cases in nature when the action–reaction symmetry is broken. In particular, suitable conditions for this are realized in colloidal suspensions and complex plasmas. Since the first theories and simulations of the nonreciprocal effective interactions between microparticles in complex plasmas were published in 1995–1996, there have been hundreds of studies in the theoretical development of this theme. However, despite such a rich theoretical background, one of the important unsolved problems is a direct experimental determination of the nonreciprocal interparticle interaction forces. Here, we studied experimentally in detail the forces of the nonreciprocal effective interaction between microparticles suspended a radio-frequency produced plasma sheath. For this purpose, an experimental method based on an analysis of the spectral density of random processes in an open dissipative two-particle system was developed. In contrast to previous investigations, the proposed method takes into account random and dissipative processes in the system, does not require a special design of the experimental setup and any external perturbations, pre-measurements of external fields and any assumptions about the type of interaction. We found that even small charge changes of one particle, caused by its thermal motion in a wake field of another particle, can lead to a significant change in the effective (measurable) interaction between the particles.

Characterization and origins of cell-free mitochondria in healthy murine and human blood

Intact cell-free mitochondria have been reported in microparticles (MPs) in murine and human bodily fluids under disease conditions. However, cellular origins of circulating extracellular mitochondria have not been characterized. We hypothesize that intact, cell-free mitochondria from heterogeneous cellular sources are present in the circulation under physiological conditions. To test this, circulating MPs were analyzed using flow cytometry and proteomics. Murine and human platelet-depleted plasma showed a cluster of MPs positive for the mitochondrial probe MitoTracker. Transgenic mice expressing mitochondrial-GFP showed GFP positivity in plasma MPs. Murine and human mitochondria-containing MPs were positive for the platelet marker CD41 and the endothelial cell marker CD144, while hematopoietic CD45 labeling was low. Both murine and human circulating cell-free mitochondria maintained a transmembrane potential. Circulating mitochondria were able to enter rho-zero cells, and were visualized using immunoelectron microscopic imaging. Proteomics analysis identified mitochondria specific and extracellular vesicle associated proteins in sorted circulating cell-free human mitochondria. Together the data provide multiple lines of evidence that intact and functional mitochondria originating from several cell types are present in the blood circulation.

Cell-Derived Microparticles and Acute Coronary Syndromes: Is there a Predictive Role for Microparticles?

Despite the recent advances in the treatment of Acute Coronary Syndromes (ACS), patients with ACS are still exposed to an increased risk for adverse cardiovascular events, while their prognosis is difficult to determine. Experimental and clinical studies have shown that cell-derived Microparticles (MPs) are associated with the underlying pathophysiological processes that are responsible for atherogenesis and may be causally implicated in the induction of atherothrombosis. In the present article, we aimed to review the available evidence regarding the predictive role of MPs in patients with ACS. Evidence suggests that endothelial MPs are associated with future adverse cardiovascular events in patients with ACS. Platelet-derived MPs have been excessively studied, since they have been found to trigger the coagulation cascade; however, their role as predictors of future cardiovascular events remains debatable. The role of red blood cell-derived MPs is more intriguing; they have been proposed as markers of ongoing thrombosis in patients with ACS, while previous studies have shown that they have anti-coagulant properties in healthy individuals. Leukocyte-derived MPs may also have a predictive role, although the studies regarding these are still limited. Last but not least, it was an interesting discovery that circulating MPs can provide information regarding the angiographic lesions in patients with ACS. The concept of MPs as potential circulating biomarkers in patients with ACS holds much promise. However, large-scale clinical studies are required to evaluate whether the measurement of plasma MPs could be of clinical significance and, thus, dictate a more aggressive treatment strategy in patients with high levels of circulating MPs.

Oxidized Low-Density Lipoprotein Induces Macrophage Production of Prothrombotic Microparticles.

BackgroundActivated vascular cells produce submicron prothrombotic and proinflammatory microparticle vesicles. Atherosclerotic plaques contain high levels of microparticles. Plasma microparticle levels increase during acute coronary syndromes and the thrombotic consequences of plaque rupture likely involve macrophage‐derived microparticles (MΦMPs). The activation pathways that promote MΦMP production remain poorly defined. This study tested the hypothesis that signals implicated in atherogenesis also stimulate MΦMP production.Methods and ResultsWe stimulated human primary MΦs with proinflammatory cytokines and atherogenic lipids, and measured MΦMP production by flow cytometry. Oxidized low‐density lipoprotein (oxLDL; 25 µg/mL) induced MΦMP production in a concentration‐dependent manner (293% increase; P<0.001), and these oxLDL MΦMP stimulatory effects were mediated by CD36. OxLDL stimulation increased MΦMP tissue factor content by 78% (P<0.05), and oxLDL‐induced MΦMP production correlated with activation of caspase 3/7 signaling pathways. Salvionolic acid B, a CD36 inhibitor and a CD36 inhibitor antibody reduced oxLDL‐induced MΦMP by 67% and 60%, respectively. Caspase 3/7 inhibition reduced MΦMP release by 52% (P<0.01) and caspase 3/7 activation increased MΦMP production by 208% (P<0.01). Mevastatin pretreatment (10 µM) decreased oxLDL‐induced caspase 3/7 activation and attenuated oxLDL‐stimulated MΦMP production and tissue factor content by 60% (P<0.01) and 43% (P<0.05), respectively.ConclusionsOxLDL induces the production of prothrombotic microparticles in macrophages. This process depends on caspases 3 and 7 and CD36 and is inhibited by mevastatin pretreatment. These findings link atherogenic signaling pathways, inflammation, and plaque thrombogenicity and identify a novel potential mechanism for antithrombotic effects of statins independent of LDL lowering.

Plasma microparticles of sickle patients during crisis or taking hydroxyurea modify endothelium inflammatory properties

AbstractMicroparticles (MPs) are submicron extracellular vesicles exposing phosphatidylserine (PS), detected at high concentration in the circulation of sickle cell anemia (SS) patients. Several groups studied the biological effects of MPs generated ex vivo. Here, we analyzed for the first time the impact of circulating MPs on endothelial cells (ECs) from 60 sickle cell disease (SCD) patients. MPs were collected from SCD patients and compared with MPs isolated from healthy individuals (AA). Other plasma MPs were purified from SS patients before and 2 years after the onset of hydroxyurea (HU) treatment or during a vaso-occlusive crisis and at steady-state. Compared with AA MPs, SS MPs increased EC ICAM-1 messenger RNA and protein levels, as well as neutrophil adhesion. We showed that ICAM-1 overexpression was primarily caused by MPs derived from erythrocytes, rather than from platelets, and that it was abolished by MP PS capping using annexin V. MPs from SS patients treated with HU were less efficient to induce a proinflammatory phenotype in ECs compared with MPs collected before therapy. In contrast, MPs released during crisis increased ICAM-1 and neutrophil adhesion levels, in a PS-dependent manner, compared with MPs collected at steady-state. Furthermore, neutrophil adhesion was abolished by a blocking anti–ICAM-1 antibody. Our study provides evidence that MPs play a key role in SCD pathophysiology by triggering a proinflammatory phenotype of ECs. We also uncover a new mode of action for HU and identify potential therapeutics: annexin V and anti–ICAM-1 antibodies.

High concentration of the plasma microparticles for venous thromboembolism associated with lung cancer.

Limited biomarkers are used for predicting risk of venous thromboembolism (VTE) associated with cancer. Circulating microparticles (MPs), especially tissue factor- positive microparticles (TF+MPs), play an important role in the development of cancer-associated VTE. This study investigated the predictive value of plasma MPs and TF+MPs for VTE in lung cancer. A case-control study was performed using the Beijing Chao-Yang Hospital Lung Cancer Registry. Cases had VTE occurring 3months before or after a diagnosis of lung cancer. Controls were patients with lung cancer without VTE matched for age, histology and stage. The proportion of MPs and TF+MPs was evaluated by light-scatter-based flow cytometry. Between January 2012 and December 2015, 30 cases with VTE and 60 controls without VTE were included. The proportion of MPs and TF+MPs was significantly more elevated in patients with VTE than in those without VTE (P<0.05 for both). By multivariate logistic regression analysis, MPs (OR 1.153; 95% CI 1.068-1.245; P<0.001) and adenocarcinoma (OR 3.223; 95% CI 1.062-9.782; P=0.039) were significantly associated with VTE. The sensitivity of the proportion of MPs in diagnosing VTE was 93.3%, and the specificity was 70.0%. The sensitivity of the proportion of TF+MPs in diagnosing VTE was 66.7%, and the specificity was 88.3%. The area under the receiver operating characteristic curves for the diagnostic of the proportion of MPs and TF+MPs values were 0.836 (95% CI 0.750-0.922, P<0.001) and 0.828 (95% CI 0.736-0.920, P<0.001) respectively. The elevated proportion of MPs and TF+MPs might help predict VTE associated with lung cancer.

Characterization of cell-derived microparticles in synovial fluid and plasma of patients with rheumatoid arthritis

Microparticles (MP) are proposed to play a role in the pathogenesis of rheumatoid arthritis (RA). This study aimed to profile cell lineage-specific MP in patients with RA, osteoarthritis (OA), and healthy controls (HC) in synovial fluid and circulation. Patients with RA (n = 40), OA (n = 30) and HC (n = 33) were included. Cell-free synovial fluid (SF) and platelet-poor plasma samples were stained with annexin V APC and antibodies against CD45, CD20, CD14, CD4, CD8, CD66b, and CD61 for multicolor flow cytometry. Mann-Whitney U test/unpaired T test was used to assess intergroup differences among RA and OA SF and clinical, serological phenotypes of RA based on normality distribution; Kruskal-Wallis test with Dunn's multiple comparisons for comparing plasma MPs among RA, OA, and HC. Correlation between MP proportions and disease parameters was assessed by Spearman's correlation. The proportion of annexin V+ MP in SF of patients with RA [5 (6.35)] [median (IQR)] was higher compared to OA [1.8 (1.35), p < 0.001] and plasma of patients with RA [3.45 (5.63)] compared to OA [1.85 (1.4)] and HC [0.9 (1.1), p < 0.001]. Leukocyte-derived [0.85 (1.17)], granulocyte-derived [0.4 (2.05)], monocyte-derived [0.4 (0.4)], and T cell-derived MP [CD4+ -0.1 (0.1); CD8+ -0.1(0.1)] were higher in RA SF (p < 0.001). Platelet-derived MP (PMP) were the major fraction [1.5 (4.23), p < 0.001] in RA plasma. Leukocyte-derived MP were higher in RA plasma [0.1 (0.2); p < 0.001) than OA and HC. Annexin V+ MP and PMP were higher in the SF of RA with extra-articular manifestations (n = 15), as compared to those without (n = 25) (p = 0.02; p < 0.01, respectively). High SF granulocyte-derived MP were observed in patients with established RA (n = 24), ACPA-positive RA (n = 32) compared to their negative counterparts (p = 0.03; p = 0.02, respectively). Our observations of higher proportions of cell-derived MP in the plasma and synovial fluid of DMARD-naïve RA patients, their clinical and serological phenotypes suggest their role in dynamic cross talk between the joint and systemic circulation, disease pathology, and progression.

High-precision in-situ size measurements of single microparticles in an RF plasma

An in-situ method to measure the radii of single microparticles in plasmas with high precision is presented. The particles are trapped in the plasma sheath and illuminated with laser light. Using out-of-focus imaging and polarizing optics, the angle- and polarization-resolved scattering intensities are measured and compared to Lorenz-Mie theory. A two-stage fit procedure is introduced to obtain the complex refractive index in addition to the particle radius. Complementary long-distance microscopy measurements are performed to compare with fit results. The method is applied to particles of different materials used in complex plasma research to measure etch rates due to plasma inherent processes.

Microparticles from vascular endothelial growth factor pathway inhibitor-treated cancer patients mediate endothelial cell injury.

Vascular endothelial growth factor pathway inhibitors (VEGFi), used as anti-angiogenic drugs to treat cancer are associated with cardiovascular toxicities through unknown molecular mechanisms. Endothelial cell-derived microparticles (ECMPs) are biomarkers of endothelial injury and are also functionally active since they influence downstream target cell signalling and function. We questioned whether microparticle (MP) status is altered in cancer patients treated with VEGFi and whether they influence endothelial cell function associated with vascular dysfunction. Plasma MPs were isolated from cancer patients before and after treatment with VEGFi (pazopanib, sunitinib, or sorafenib). Human aortic endothelial cells (HAECs) were stimulated with isolated MPs (106 MPs/mL). Microparticle characterization was assessed by flow cytometry. Patients treated with VEGFi had significantly increased levels of plasma ECMP. Endothelial cells exposed to post-VEGFi treatment ECMPs induced an increase in pre-pro-ET-1 mRNA expression, corroborating the increase in endothelin-1 (ET-1) production in HAEC stimulated with vatalanib (VEGFi). Post-VEGFi treatment MPs increased generation of reactive oxygen species in HAEC, effects attenuated by ETA (BQ123) and ETB (BQ788) receptor blockers. VEGFi post-treatment MPs also increased phosphorylation of the inhibitory site of endothelial nitric oxide synthase (eNOS), decreased nitric oxide (NO), and increased ONOO− levels in HAEC, responses inhibited by ETB receptor blockade. Additionally, gene expression of proinflammatory mediators was increased in HAEC exposed to post-treatment MPs, effects inhibited by BQ123 and BQ788. Our findings define novel molecular mechanism involving interplay between microparticles, the ET-1 system and endothelial cell pro-inflammatory and redox signalling, which may be important in cardiovascular toxicity and hypertension associated with VEGFi anti-cancer treatment. New and noteworthy: our novel data identify MPs as biomarkers of VEGFi-induced endothelial injury and important mediators of ET-1-sensitive redox-regulated pro-inflammatory signalling in effector endothelial cells, processes that may contribute to cardiovascular toxicity in VEGFi-treated cancer patients.

Increased circulating microparticles in streptozotocin-induced diabetes propagate inflammation contributing to microvascular dysfunction.

Circulating microparticles (MPs) are elevated in many cardiovascular diseases and have been considered as biomarkers of disease prognosis; however, current knowledge of MP functions has been mainly derived from in vitro studies and their precise impact on vascular inflammation and disease progression remains obscure. Using a diabetic rat model, we identified a >130-fold increase in MPs in plasma of diabetic rats compared to normal rats, the majority of which circulated as aggregates, expressing multiple cell markers and largely externalized phosphatidylserine; vascular images illustrate MP biogenesis and their manifestations in microvessels of diabetic rats. Using combined single microvessel perfusion and systemic cross-transfusion approaches, we delineated how diabetic MPs propagate inflammation in the vasculature and transform normal microvessels into an inflammatory phenotype observed in the microvessels of diabetic rats. Our observations derived from animal studies resembling conditions in diabetic patients, providing a mechanistic insight into MP-mediated pathogenesis of diabetes-associated multi-organ microvascular dysfunction. In various cardiovascular diseases, microparticles (MPs), the membrane-derived vesicles released during cell activation, are markedly increased in the circulation. These MPs have been recognized to play diverse roles in the regulation of cellular functions. However, current knowledge of MP function has been largely derived from in vitro studies. The precise impact of disease-induced MPs on vascular inflammation and disease progression remains obscure. In this study we investigated the biogenesis, profile and functional roles of circulating MPs using a streptozotocin-induced diabetic rat model with well-characterized microvascular functions. Our study revealed a >130-fold increase in MPs in the plasma of diabetic rats compared to normal rats. The majority of these MPs originate from platelets, leukocytes and endothelial cells (ECs), and circulate as aggregates. Diabetic MPs show greater externalized phosphatidylserine (PS) than normal MPs. When diabetic plasma or isolated diabetic MPs were perfused into normal microvessels or systemically transfused into normal rats, MPs immediately adhered to endothelium and subsequently mediated leukocyte adhesion. These microvessels then exhibited augmented permeability responses to inflammatory mediators, replicating the microvascular manifestations observed in diabetic rats. These effects were abrogated when MPs were removed from diabetic plasma or when diabetic MPs were pre-coated with a lipid-binding protein, annexinV, suggesting externalized PS to be key in mediating MP interactions with endothelium and leukocytes. Our study demonstrated that the elevated MPs in diabetic plasma are actively involved in the propagation of vascular inflammation through their adhesive surfaces, providing mechanistic insight into the pathogenesis of multi-organ vascular dysfunction that commonly occurs in diabetic patients.