Microparticles Subpopulations Research Articles

Association between endothelial microparticles and objectively measured physical activity in adults with obesity

BackgroundCardiovascular disease (CVD) is a serious global public health concern that often originates from endothelial dysfunction. Microparticles (MPs) can reflect alterations in tissue or cell function phenotype and responsiveness under various physiological and pathological conditions. MPs derived from endothelial cells may serve as potential biomarkers for CVD. We investigated the association between endothelial-derived MPs (EMPs) and objectively measured physical activity (PA) in adults with obesity and their potential correlation with inflammatory and cardiometabolic biomarkers. MethodsThis cross-sectional study included 50 participants, whose daily movement behaviors were evaluated using activPAL™, which measures moderate-to-vigorous intensity PA (MVPA), light PA (LPA), and standing time. The MP sub-populations were separated from platelet-poor plasma, incubated with multiple antibodies, and analyzed using multi-color flow cytometry. Cytokines, interleukin [IL]−1β, IL-8, E-selectin, P-selectin, and intercellular adhesion molecules [ICAMs], were measured using a customized Luminex® assay kit. Pearson’s correlation coefficients were used to examine associations. ResultsMVPA was associated with lower expressions of ICAM and E-selectin in EMPs, whereas standing time was associated with a higher expression of E-selectin. A positive association exists between leukocyte-derived MP percentage and P-selectin levels in EMPs and E-selectin levels in the plasma. In addition, higher plasma P-selectin level was associated with higher P- and E-selectin levels in EMPs, as was higher inflammation (IL-1β and IL-8). ConclusionThe MPs were negatively associated with MVPA and positively associated with standing time in the participants with obesity. These results underscore the importance of promoting PA interventions, especially at moderate-to-vigorous intensities. Further longitudinal and interventional studies are warranted to elucidate the dynamic interplay among PA, MPs, and endothelial function.

Heparin Microislands in Microporous Annealed Particle Scaffolds for Accelerated Diabetic Wound Healing.

Mimicking growth factor-ECM interactions for promoting cell migration is a powerful technique to improve tissue integration with biomaterial scaffolds for the regeneration of damaged tissues. This has been attempted by scaffold-mediated controlled delivery of exogenous growth factors; however, the predetermined nature of this delivery can limit the scaffold's ability to meet each wound's unique spatiotemporal regenerative needs and presents translational hurdles. To address this limitation, we present a new approach to growth factor presentation by incorporating heparin microislands, which are spatially isolated heparin-containing microparticles that can reorganize and protect endogenous local growth factors via heterogeneous sequestration at the microscale in vitro and result in functional improvements in wound healing. More specifically, we incorporated our heparin microislands within microporous annealed particle (MAP) scaffolds, which allows facile tuning of microenvironment heterogeneity through ratiometric mixing of microparticle sub-populations. In this manuscript, we demonstrate the ability of heparin microislands to heterogeneously sequester applied growth factor and control downstream cell migration in vitro. Further, we present their ability to significantly improve wound healing outcomes (epidermal regeneration and re-vascularization) in a diabetic wound model relative to two clinically relevant controls.

Elevated Circulating Microparticle Subpopulations in Incidental Cerebral White Matter Hyperintensities: A Multimodal Study.

Asymptomatic (or “silent”) manifestations of cerebral small vessel disease (CSVD) are widely recognized through incidental findings of white matter hyperintensities (WMHs) as a result of magnetic resonance imaging (MRI). This study aims to examine the potential associations of surrogate markers for the evaluation of white matter integrity in CSVD among asymptomatic individuals through a battery of profiling involving QRISK2 cardiocerebrovascular risk prediction, neuroimaging, neurocognitive evaluation, and microparticles (MPs) titers. Sixty asymptomatic subjects (mean age: 39.83 ± 11.50 years) with low to moderate QRISK2 scores were recruited and underwent neurocognitive evaluation for memory and cognitive performance, peripheral venous blood collection for enumeration of selected MPs subpopulations, and 3T MRI brain scan with specific diffusion MRI (dMRI) sequences inclusive of diffusion tensor imaging (DTI). WMHs were detected in 20 subjects (33%). Older subjects (mean age: 46.00 ± 12.00 years) had higher WMHs prevalence, associated with higher QRISK2 score and reduced processing speed. They also had significantly higher mean percentage of platelet (CD62P)- and leukocyte (CD62L)-derived MPs. No association was found between reduced white matter integrity—especially at the left superior longitudinal fasciculus (LSLF)—with age and neurocognitive function; however, LSLF was associated with higher QRISK2 score, total MPs, and CD62L- and endothelial cell-derived MPs (CD146). Therefore, this study establishes these multimodal associations as potential surrogate markers for “silent” CSVD manifestations in the well-characterized cardiocerebrovascular demographic of relatively young, neurologically asymptomatic adults. Furthermore, to the best of our knowledge, this study is the first to exhibit elevated MP counts in asymptomatic CSVD (i.e., CD62P and CD62L), which warrants further delineation.

Circulating microparticle subpopulation in metabolic syndrome: relation to oxidative stress and coagulation markers.

BackgroundCirculating microparticles (MPs) contribute to the pathogenesis of atherothrombotic disorders and are raised in cardiovascular diseases. Herein, we aimed to investigate the effect of moderate metabolic abnormalities in an early stage of metabolic syndrome (MetS) on the level of MP subpopulations and to study relationships between MP subpopulations and both oxidative stress and coagulation markers.MethodsFlow cytometry used to evaluate circulating MPs subpopulations in 40 patients with an early stage MetS and 30 healthy controls. ELISA was used to quantify plasminogen activator inhibitor type 1/tissue plasminogen activator (PAI-1/TPA) while plasma glutathione peroxidase (GPx) activity was measured spectrophotometrically.ResultsTotal MPs were significantly elevated in MetS (P<0.001). Glutathione peroxidase and PAI1/TPA activity was significantly increased in subjects with MetS (P<0.001). Waist circumference, diastolic blood pressure, and total cholesterol positively influenced levels of total MPs, platelet-derived microparticles, and endothelium-derived microparticles. Fasting blood glucose, cholesterol, triglycerides, and low-density lipoprotein positively influenced the coagulation factors (TPA, PAI1). However, high-density lipoprotein negatively influenced platelet-derived MPs and factors associated with fibrinolysis (TPA, PAI1).ConclusionElevated circulating MPs are associated with MetS abnormalities, oxidative stress and coagulation factors and may act as early predictor of metabolic syndrome with risk of cardiovascular disease.

Circulating microparticle subpopulations in systemic lupus erythematosus are affected by disease activity

BackgroundImmune cells under chronic inflammation shed microparticles (MPs) that could fuel the inflammatory responses and atherosclerosis typically presented in systemic lupus erythematosus (SLE). This study analyzes total and subset-specific MPs from SLE patients and their possible influence on clinical features, leukocyte activation and serum cytokines. MethodsTotal MPs and those derived from platelets, endothelial cells, monocytes, granulocytes and T-cells were quantified in plasma of 106 SLE patients and 33 healthy controls by flow cytometry. MP amounts were analyzed according to clinical manifestations, blood leukocyte populations and circulating cytokines (IFNα, TNFα, IL-10, BLyS, IL-17, IL-1β, CXCL10, CCL-2, CCL3, leptin). Finally, the in vitro effect of SLE-isolated MPs on the leukocyte activation status was analyzed. ResultsTotal circulating MPs in SLE patients were related to increased disease duration and the presence of cardiovascular disease. Furthermore, patients displayed increased counts of MPs from platelets, monocytes and T-lymphocytes, especially in SLE patients with disease activity or with TNFαhigh-profile. Accordingly, MPs were associated with increased expression of activation markers in blood T-cells and monocytes. Finally, analyses propose a role of glucocorticoids in MPs generation and leukocyte activation since both fresh and cultured T-cells under this treatment presented higher IL-10 and CD25 production. ConclusionsThe altered profile of subset-specific SLE-MPs was influenced by the disease activity and altered status of leukocyte native cells, also associated with a TNFαhigh-profile. Translational resultsSLE patients, especially those with disease activity, displayed increased counts of MPs derived from platelets, monocytes and T-lymphocytes, which were more frequently found in TNFαhigh-type patients. The origin of such SLE-MP subsets seems to be related to the over-activated status of T-cells and monocytes characteristic of these patients. Ex vivo and in vitro analyses propose a role of glucocorticoids in the generation of circulating MPs and leukocyte activation in SLE patients.

Microparticle subpopulations are potential markers of disease progression and vascular dysfunction across a spectrum of connective tissue disease

ObjectiveMicroparticles (MPs) are membrane-bound vesicles derived from vascular and intravascular cells such as endothelial cells (EMPs) and platelets (PMPs). We investigated EMP and PMP numbers across a spectrum of autoimmune rheumatic diseases (AIRDs) with the aim of comparing the levels of, and relationship between, EMPs and PMPs. MethodsPatients with Systemic Lupus Erythematosus (SLE) (n=24), Systemic Sclerosis (SSc) (n=24), Primary Raynauds Phenomenon (RP) (n=17) and “other CTD” (n=15) (Primary Sjogrens Syndrome, UCTD or MCTD) as well as 15 healthy controls were recruited. EMPs and PMPs were quantified using flow cytometry. Associations between MP levels and objective functional vascular assessments were evaluated. ResultsSLE patients had significantly higher EMPs compared with healthy controls and SSc patients. Higher PMP levels were noted in SSc and primary RP when compared to healthy controls and ‘other CTD’ patients. A modest correlation was noted between EMP and PMP levels in healthy controls (Spearman r=0.6, p=0.017). This relationship appeared stronger in SLE (r=0.72, p<0.0001) and other CTD patients (r=0.75, p<0.0001). The association between EMPs and PMPs was notably less strong in SSc (r=0.45, p=0.014) and RP (r=0.37, p=0.15). A significantly lower EMP/PMP ratio was detected in SSc/RP patients in comparison to both healthy controls and SLE/other CTD patients. Higher EMP and PMP levels were associated with higher digital perfusion following cold challenge in SSc. In contrast, higher PMP (but not EMP) levels were associated with lower digital perfusion at both baseline and following cold challenge in primary RP. Higher PMP levels were associated with greater endothelial-independent dilation in patients with SLE. ConclusionMP populations differ across the spectrum of AIRDS, possibly reflecting differences in vascular cell injury and activation. MP levels are associated with functional assessments of vascular function and might have a role as novel vascular biomarkers in AIRDs. Significance and innovationsLevels of circulating endothelial and platelet microparticles differ between SSc/primary RP compared with SLE and other CTDs (UCTD, MCTD and Primary Sjogrens). MP release may occur within different vascular sites across these disease groups (macrovascular and microvascular).The association between circulating MP levels and objective assessment of macro- and microvascular dysfunction within these disease areas suggests that MPs might have a useful role as novel circulating biomarkers of vascular disease within the CTDs.

Circulating cell-derived microparticles as biomarkers in cardiovascular disease.

Cardiovascular diseases (CVDs) are a common cause of death, and a search for biomarkers for risk stratification is warranted. Elevated levels of cell-derived microparticles (MPs) are found in patients with CVD and in groups with risk factors for CVD. Subpopulations of MPs are promising biomarkers for improving risk prediction, as well as monitoring treatment. However, the field has been hampered by technical difficulties, and the ongoing development of sensitive standardized techniques is crucial for implementing MP analyses in the clinic. Large prospective studies are required to establish which MPs are of prognostic value in different patient groups. In this review, we discuss methodological challenges and progress in the field, as well as MP populations that are of interest for further clinical evaluation.

Development of a NanoBioAnalytical platform for "on-chip" qualification and quantification of platelet-derived microparticles

Blood microparticles (MPs) are small membrane vesicles (50–1000nm), derived from different cell types. They are known to play important roles in various biological processes and also recognized as potential biomarkers of various health disorders. Different methods are currently used for the detection and characterization of MPs, but none of these methods is capable to quantify and qualify total MPs at the same time, hence, there is a need to develop a new approach for simultaneous detection, characterization and quantification of microparticles. Here we show the potential of surface plasmon resonance (SPR) method coupled to atomic force microscopy (AFM) to quantify and qualify platelet-derived microparticles (PMPs), on the whole nano-to micro-meter scale. The different subpopulations of microparticles could be determined via their capture onto the surface using specific ligands. In order to verify the correlation between the capture level and the microparticles concentration in solution, two calibration standards were used: Virus-Like Particles (VLPs) and synthetic beads with a mean diameter of 53nm and 920nm respectively. The AFM analysis of the biochip surface allowed metrological analysis of captured PMPs and revealed that more than 95% of PMPs were smaller than 300nm. Our results suggest that our NanoBioAnalytical platform, combining SPR and AFM, is a suitable method for a sensitive, reproducible, label-free characterization and quantification of MPs over a wide concentration range (≈107 to 1012 particles/mL; with a limit of detection (LOD) in the lowest ng/µL range) which matches with their typical concentrations in blood.

A new cytofluorimetric approach to evaluate the circulating microparticles in subjects with antiphospholipid antibodies

IntroductionGrowing evidence supports the idea that microparticles (MPs) could contribute to the pathogenesis of the thrombotic phenomena associated with antiphospholipid antibody syndrome (APS), inducing a hypercoagulable state. But, to date, different approaches to evaluate circulating MPs and conflicting results have been reported. Materials and methodsWe have characterized the different circulating subpopulations of MPs in APS patients, and in asymptomatic aPL-positive subjects (carriers) by examining the correlation between the amount and phenotype of MPs and the clinical parameters. Forty-eight subjects were enrolled: 16 with primary APS, 16 aPL-positive, but without clinical criteria for APS (carriers), and 16 healthy subjects. The levels of MPs were evaluated using a new cytofluorimetric approach based on BD Horizon Violet Proliferation dye (VPD) 450. Results and conclusionsUsing a new detection cytofluorimetric approach, we demonstrated that the AnnV-negative MPs, underestimated/or excluded in the previous studies, are a large subset of circulating MPs. Also, the levels of MPs in the plasma of aPL positive subjects indicate a state of cellular activation, which is much more pronounced in patients with APS compared to aPL carriers. Moreover, the preliminary data of our pilot study suggest that the evaluation of circulating MPs, in particular PMPs and EMPs, could be used as a surrogate biomarker for platelet and vascular damage monitoring and, if confirmed in a more numerous cohort of patients, it could be used as a prognostic factor to identify aPL positive subjects at higher risk of developing thrombosis.

Increased monocyte/neutrophil and pro-coagulant microparticle levels and overexpression of aortic endothelial caveolin-1β in dyslipidemic sand rat, Psammomys obesus

AimsTo compare the effects of a high-energy diet (HED) with those of a low-energy diet (LED) on biochemical parameters, microparticle (MP) subpopulations and endothelial caveolin-1 (cav-1) protein expression in Psammomys obesus (P. obesus). MethodsAfter 12weeks of feeding with either the HED or LED, fasting plasma glucose and lipid parameters were measured using an enzymatic colorimetric kit while serum insulin concentration was determined with radioimmunoassay kits. MP subpopulations and cav-1 protein expression were quantified using flow cytometry and western blot analysis, respectively. ResultsWe observed that the HED caused a marked increase in lipid parameters, even in normoglycemic P. obesus. The total number of circulating MPs and the numbers of platelet-, leukocyte-, and erythrocyte-derived MPs were unaltered in the HED group. However, the HED induced increases in the numbers of monocytes/neutrophils and procoagulant MPs and a decrease in the endothelial MP levels. Cav-1β protein expression and reactive oxygen species production were increased in the vascular endothelium of HED-treated P. obesus. ConclusionFrom these findings, it is indicated that the HED exerts deleterious effects on the vascular system by increasing the monocyte/neutrophil and procoagulant MP levels, which may lead to cav-1β protein overexpression in dyslipidemic P. obesus.

Effect of Acute Endurance Exercise on Circulating Angiogenic Cell and Microparticle Subpopulations

Effect of Acute Endurance Exercise on Circulating Angiogenic Cell and Microparticle Subpopulations

Improved endothelial function and decreased levels of endothelium-derived microparticles after transcatheter aortic valve implantation.

Degenerative aortic valve stenosis (AVS) is independently associated with endothelial dysfunction and increased levels of circulating endothelium-derived microparticles (EMPs) as a marker of compromised endothelial integrity. The aim of this study was to investigate whether therapy for severe AVS by transcatheter aortic valve implantation (TAVI) improves endothelial function and decreases EMPs. Fifty-six patients with indication for TAVI due to symptomatic severe AVS were prospectively enrolled. Brachial wall shear stress (WSS), endothelial function and circulating microparticles (MPs) were measured before and three months following TAVI. Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound. MP subpopulations were discriminated by flow cytometry according to the expression of established surface antigens: CD31+/CD41-, CD144+ and CD62E+ as EMPs and CD41+ as platelet-derived MPs (PMPs). In patients with severe AVS, decreased brachial WSS was an independent predictor of low FMD. At three-month follow-up after TAVI, WSS and FMD increased along with decreased levels of EMPs as compared to pre TAVI. Decrease of CD31+/CD41-, CD144+ and CD62E+ EMP levels correlated with the increase of FMD. Therapy for AVS by TAVI was associated with improved endothelial function and integrity indicating beneficial effects of TAVI on systemic arterial function.

Formation of microparticles in the injured brain of patients with severe isolated traumatic brain injury.

The potential pathophysiological role of circulating microparticles (MPs) has been recognized in various conditions, such as cardiovascular and thrombotic diseases. Traumatic brain injury (TBI) has a complex pathophysiology that involves coagulopathy and inflammation. We investigated endothelial-, platelet-, and leukocyte-derived microparticles (EMPs, PMPs, and LMPs, respectively) in 16 patients with severe isolated TBI. Arterial and cerebrovenous samples were taken repeatedly, during 1-72 h after injury. Subpopulations of MPs, exposing tissue factor (TF) and P-selection, were also studied. MP counts in cerebrovenous samples, irrespective of cellular origin, were higher in TBI cases, compared to healthy controls (peak levels of EMPs were approximately 7 times higher, PMPs 1.4 times higher, and LMPs 2 times higher, respectively; p<0.001 for all). MP counts declined sharply from high levels shortly after the trauma toward slightly elevated levels 72 h later. EMPs and PMPs exposing TF, as well as PMPs exposing P-selection, showed a transcranial gradient with higher concentration in cerebrovenous, compared to arterial, samples. In contrast, LMPs exposing TF were higher in arterial samples, suggesting accumulation of LMPs in the brain. We conclude that the pattern of circulating MPs is altered after TBI. PMPs exposing P-selection and EMPs exposing TF seem to be generated in the injured brain, whereas LMPs exposing TF are accumulated. The pathophysiological significance of these changes in MP pattern in TBI should be further investigated. Including MPs exposing brain-specific antigens in the assessment of brain injury would give further information of origin and likely give additional information of the size of the injury, given that the MP phenotypes investigated in the present study are not brain-specific markers.

C0344: Characterization of Microparticle Subpopulations in Cancer Patients with Thrombosis

C0344: Characterization of Microparticle Subpopulations in Cancer Patients with Thrombosis

A flow cytometric method for characterization of circulating cell-derived microparticles in plasma

Background and aimPrevious studies on circulating microparticles (MPs) indicate that the majority of MPs are of a size below the detection limit of most standard flow cytometers. The objective of the present study was to establish a method to analyze MP subpopulations above the threshold of detection of a new generation BD FACSAria™ III digital flow cytometer.MethodsWe analyzed MP subpopulations in plasma from 24 healthy individuals (9 males and 15 females). MPs were identified according to their size (<1.0-µm), by Lactadherin-FITC labelling, and by exposure of cell-specific markers. The sensitivity of the flow cytometer was tested against that of a previous-generation instrument FC500. Reproducibility of the FACSAria and our set-up was investigated, and the percentage of phosphatidylserine (PS) exposing MPs binding Lactadherin was determined.ResultsBy using a flow cytometric approach we identified and quantitated MPs derived from platelets, monocytes, erythrocytes and endothelial cells. In addition, levels of tissue factor-positive MPs were determined. The FACSAria demonstrated improved sensitivity and increased MP detection range compared to the FC500 instrument. The reproducibility of PS+PMP and PS+MP measurements was 11.7 and 23.2%, respectively. When expressed as a percentage of total MPs, the PS-positive MP population represented 15.1±5.5%, and PS-positive MPs were significantly increased in men.ConclusionWe have established a method to measure MPs above the detection limit of a new generation flow cytometer and derived from a number of cell-types in a healthy population of men and women.

Improved endothelial dysfunction and decreased endothelial injury after transaortic valve implacement

Purpose: Degenerative aortic valve stenosis is independently associated with endothelial dysfunction and increased endothelial injury. Circulating endothelial derived microparticles (EMPs) have been shown to be a marker of endothelial injury and are established as a prognostic marker of cardiovascular outcome. Therefore, we tested in patients with aortic valve stenosis, whether resolvement of the aortic valve stenosis improves endothelial dysfunction and decreases endothelial injury, as assessed by levels of EMPs. Methods: Endothelial function and circulating microparticles (MPs) were measured at a 3 month study period without transaortic valve implacement (TAVI) compared to a 3 month study period after TAVI. Of 81 patients included in the study, n=56 could be followed up completely. Measurements of endothelial function and number of circulating MPs were taken before and after each study period: Endothelial function was assessed as flow-mediated dilation (FMD) using ultrasound. MP subpopulations were discriminated by flow cytometry according to the expression of established surface antigens. The MP subpopulations were defined as follow: CD31+/CD41-, CD 144+ and CD 62E+ as EMPs, CD41+ as platelet-derived MPs (PMPs), and CD45+ as leucocytes derived MPs (LMPs). Results: Endothelial function increased and vascular injury (levels of EMPs) decreased at 3 month follow up after TAVI. Levels of LMPs also decreased. Levels of total MPs and PMPs did not change. Conclusion: Endothelial dysfunction improves and endothelial inury decreases after resolvement of the aortic valve stenosis by TAVI. Our study complements previously shown association indicating that aortic valve stenosis independently impairs endothelial function.

The LPS‐induced increase in circulating microparticles is not affected by vitamin C in humans

Microparticles (MP) are considered to promote coagulation. This study aimed to characterize the time course of MP levels and the effect of high-dose vitamin C on MP formation during inflammation in an in vivo Escherichia coli endotoxin (LPS) model. Microparticle formation was studied in 14 male subjects in a cross-over trial who received either intravenous vitamin C at 320 mg/kg body weight (BW) or 480 mg/kg BW or saline solution in a random order on alternate trial days 3 h after intravenous exposure to LPS (2 ng/kg BW). Venous blood samples were taken before, 3 and 6 h after LPS. D-dimer, leucocyte count, C-reactive protein, plasma vitamin C and body temperature were assessed as inflammatory parameters. MP were detected using flow cytometric analysis and expressed in 10³ MP/mL plasma. Microparticles levels were decreased from baseline 848 units [range 431-1705] by 21% to 671 units [253-1586] at 3 h and increased by 32% to 1119 units [288-4443] at 6 h after LPS. This pattern was not influenced by administration of vitamin C, with a change from 730 units [399-1396] at baseline by an increase to 832 units [215-2168] at 3 h to 1055 units [350-4858] at 6 h. MP subpopulations followed similar dynamics. Alterations in inflammatory parameters were independent from vitamin C administration during endotoxemia. Microparticles are increased in acute systemic inflammation with inconsistent changes in MP subgroups in healthy subjects. Systemic vitamin C administration does not mitigate MP formation and D-dimer levels during acute systemic inflammation, suggesting that MP-induced coagulation activity is not affected by vitamin C.

Microparticles: New Protagonists in Pericellular and Intravascular Proteolysis

Microparticles (MPs) are small vesicles resulting from the shedding of cellular membrane during activation or apoptosis processes. Beyond their well-described procoagulant property, accumulating data show that specific endothelial cell-, leukocyte-, tumor-derived MPs bind plasminogen and vectorize plasminogen activators, leading to an efficient plasmin generation and matrix metalloproteinases activation. This review focuses on the molecular equipment of MPs subpopulations that identify MPs as efficient support for plasmin generation and the potential consequences of this new function. By the combined facts that MPs may disseminate, concentrate active proteolytic molecules and represent a protective environment against soluble inhibitors, MPs behave as an efficient catalytic surface involved in vascular and matrix proteolysis-related biological processes. The existence of this proteolytic MPs in the circulation or in body fluids raises the question about the physiological relevance of this activity. Consequences are suggested in many biological processes such as fibrinolysis, cell survival, matrix remodeling, angiogenesis, and tumor metastasis. However, further studies will be necessary to determine the extent in which in vivo MPs contribute to these pathophysiological mechanisms and how this circulating property of MPs may represent a new biomarker in specific clinical situations.

A standardized multi-colour flow cytometry approach to characterize the many faces of peripheral circulating microparticles

Microparticles (MP) are small vescicles (<1 µm of diameter) released by several cell types and characterized by an integral plasma membrane expressing the phenotype of the cell from which they originate (Jayachandran et al., 2012). MP play a crucial role in a multitude of pathologies, Including inflammatory and autoimmune disease, diabetes, atherosclerosis, malignancies and cardiovascular disease. The role, as potential biomarker, attributed to circulating MP has been emphasized by the recent literature. In such context, multicolour flow cytometry has great potential In the MP studies (Lanuti et al., 2012). Unfortunately, consensus guidelines on MP identification and enumeration has not been reached yet, due to their small sizes. We purpose to identify, characterize and count MP from whole blood by a seven-colors flow cytometry protocol, with the aim to standardize such method and to allow the identification of both the whole compartment and different MP subpopulations (i.e. endothelial-, platelet- and leukocyte-derived MP). We optimized a novel flow cytometry protocol, using peripheral whole blood stained by a combinations of seven different antigens/probes. Different panel combinations, anticoagulants and storage conditions were evaluated to set the best protocol with the aim to obtain reliable and reproducible MP counts. MP enumeration was carried out by a single platform method by using TrueCount (BD Biosciences). Results demonstrated that the application of the newly optimized flow cytometry method here described, allows to obtain high reproducibility of MP enumeration, pointing out different MP subpopulations both in healthy donors and in different patients. This method may open new routes for the monitoring of MP numbers and phenotypes in different clinical setting.

Increased CD39 Nucleotidase Activity on Microparticles from Patients with Idiopathic Pulmonary Arterial Hypertension

BackgroundIdiopathic pulmonary arterial hypertension (IPAH) is a devastating disease characterized by increased pulmonary vascular resistance, smooth muscle and endothelial cell proliferation, perivascular inflammatory infiltrates, and in situ thrombosis. Circulating intravascular ATP, ADP, AMP and adenosine activate purinergic cell signaling pathways and appear to induce many of the same pathologic processes that underlie IPAH. Extracellular dephosphorylation of ATP to ADP and AMP occurs primarily via CD39 (ENTPD1), an ectonucleotidase found on the surface of leukocytes, platelets, and endothelial cells [1]. Microparticles are micron-sized phospholipid vesicles formed from the membranes of platelets and endothelial cells. Objectives: Studies here examine whether CD39 is an important microparticle surface nucleotidase, and whether patients with IPAH have altered microparticle-bound CD39 activity that may contribute to the pathophysiology of the disease.Methodology/ Principal FindingsKinetic parameters, inhibitor blocking experiments, and immunogold labeling with electron microscopy support the role of CD39 as a major nucleotidase on the surface of microparticles. Comparison of microparticle surface CD39 expression and nucleotidase activity in 10 patients with advanced IPAH and 10 healthy controls using flow cytometry and thin layer chromatograph demonstrate the following: 1) circulating platelet (CD39+CD31+CD42b+) and endothelial (CD39+CD31+CD42b−) microparticle subpopulations in patients with IPAH show increased CD39 expression; 2) microparticle ATPase and ADPase activity in patients with IPAH is increased.Conclusions/ SignificanceWe demonstrate for the first time increased CD39 expression and function on circulating microparticles in patients with IPAH. Further research is needed to elucidate whether these findings identify an important trigger for the development of the disease, or reflect a physiologic response to IPAH.