Cellular Origin Of Microparticles Research Articles

Immunoregulatory molecule expression on extracellular microvesicles in people living with HIV.

People living with HIV (PLWH) now benefit from combined antiviral treatments that durably control viral replication. These antiretroviral treatments decrease mortality and improve quality of life in PLWH, but do not completely control the excessive non-specific activation of the immune system in PLWH. This chronic immune activation is a key element of HIV immunopathology that contributes to the pathophysiology of inflammatory comorbid conditions, such as cardiovascular disorders, cancer and autoimmune diseases. Circulating non-exosomal extracellular vesicles, also known as microparticles (MPs) are detected in these diseases and have been linked to immune activation. The objective of this study was to characterize the MPs present in PLWH and to assess their association with chronic immune activation. We performed flow cytometry for the complete phenotypic characterization of MPs from fresh plasma from PLWH and from people without HIV as the control group. The absolute number, size and cellular origin of MPs were evaluated. The immunoregulatory profile was determined by cell origin, for MPs derived from platelets (PMPs), monocytes (MMPs) and T lymphocytes (LMPs). PLWH had significantly more circulating MPs than controls, for MPs of all sizes originating from T lymphocytes, red blood cells, neutrophils, dendritic cells, B lymphocytes and endothelial cells. PMPs and MMPs were not more numerous in PLWH, but the immunoregulatory phenotypes of these MPs differed between PLWH and controls. These differences in immunoregulatory molecule expression profile were also observed for LMPs. PDL1, ICOSL, CCR5, TGFβ1, MHC classes I and II, TRAIL, CXCR4, OX40, DC-SIGN, CTLA4 and PDL2 were more strongly expressed on the surface of MPs from PLWH than on those from controls. MPs are an important element in intercellular communication, making it possible to transfer phenotypes and functions to immune cells. The significantly higher numbers of MPs expressing diverse immunomodulatory molecules in PLWH may make a major contribution to the maintenance and/or the development of immune-cell activation in these individuals.

Role of Microparticles in the Pathogenesis of Inflammatory Joint Diseases.

Rheumatoid arthritis (RA), juvenile idiopathic arthritis (JIA), ankylosing spondylitis (AS), and psoriatic arthritis (PsA) make up a group of chronic immune-mediated inflammatory diseases (IMIDs). The course of these diseases involves chronic inflammation of joints and enthesopathies, which can result in joint damage and disability. Microparticles (MPs) are a group of small spherical membranous vesicles. The structure and cellular origin of MPs, mechanisms that stimulate their secretion and the place of their production, determine their biological properties, which could become manifest in the pathogenesis of immune-mediated inflammatory diseases. Microparticles can stimulate synovitis with proinflammatory cytokines and chemokines. MPs may also contribute to the pathogenesis of rheumatic diseases by the formation of immune complexes and complement activation, pro-coagulation activity, activation of vascular endothelium cells, and stimulation of metalloproteinase production. It seems that in the future, microparticles can become a modern marker of disease activity, a response to treatment, and, possibly, they can be used in the prognosis of the course of arthritis. The knowledge of the complexity of MPs biology remains incomplete and it requires further comprehensive studies to explain how they affect the development of rheumatic diseases. This review focuses on the immunopathogenic and therapeutic role of MPs in chronic immune-mediated inflammatory joint diseases.

Endogenous microparticles drive the proinflammatory host immune response in severely injured trauma patients.

Severe trauma affects the immune system, which in its turn is associated with poor outcome. The mediators driving the immune responses in trauma are largely unknown. The aim of this study was to investigate the role of endogenous microparticles (MPs) in mediating the immune response following severe trauma. A prospective, observational substudy of the ACIT II (Activation of Coagulation and Inflammation in Trauma II) study was performed at our academic level I trauma center. Adult multiple-trauma patients with an injury severity score of 15 or higher were included between May 2012 and June 2013. Ex vivo whole-blood stimulation with lipopolysaccharide was performed on aseptically collected patient plasma containing MPs and in plasma depleted of MPs. Flow cytometry and transmission electronic microscopy were performed on plasma samples to investigate the numbers and cellular origin of MPs. Healthy individuals served as a control group. Ten trauma patients and 10 control subjects were included. Trauma patients were significantly injured with a median injury severity score of 19 (range, 17-45). Patients were neither in shock nor bleeding. On admission to the hospital, the host response to bacterial stimulation was blunted in trauma patients compared with control subjects, as reflected by decreased production of interleukin 6 (IL-6), IL-10, and tumor necrosis factor α (P < 0.001). In trauma patients, MP-positive plasma was associated with a significantly higher synthesis of IL-6 and tumor necrosis factor α compared with plasma depleted from MPs (P = 0.047 and 0.002, respectively). Compared with control subjects, the number of circulating MPs was significantly decreased in trauma patients (P = 0.009). Most MPs originated from platelets. Multiple cellular protrusions, which result in MP formation, were observed in plasma from trauma patients, but not in control subjects. On admission, trauma patients have a reduced immune response toward endotoxin challenge, which is, at least in part, mediated by MPs, which circulate in low numbers and in early stages. Most MPs originate from platelets, which indicates that these cells may be the most important source of MPs involved in initiating an inflammatory host response after injury.

Young women with polycystic ovary syndrome have raised levels of circulating annexin V-positive platelet microparticles.

Are circulating microparticles (MPs) altered in young women with polycystic ovary syndrome (PCOS)? Women with PCOS have elevated concentrations of circulating platelet-derived MPs, which exhibit increased annexin V binding and altered microRNA (miR) profiles compared with healthy volunteers. Some studies have shown that cardiovascular risk is increased in young women with PCOS but the mechanisms by which this occurs are uncertain. Circulating MPs are elevated in patients with cardiovascular disease but the characteristics of MPs in patients with PCOS are unclear. Case-control study comprising 17 women with PCOS (mean ± SD; age 31 ± 7 years, BMI 29 ± 6 kg/m(2)) and 18 healthy volunteers (age 31 ± 6 years, BMI 30 ± 6 kg/m(2)). The study was conducted in a University hospital. Nanoparticle tracking analysis (NTA) and flow cytometry (CD41 platelet, CD11b monocyte, CD144 endothelial) were used to determine MP size, concentration, cellular origin and annexin V positivity (reflecting phosphatidylserine exposure). Fatty acid analysis was performed by gas chromatography and MP miR expression profiles were compared by microarray. PCOS subjects showed increased MP concentrations compared with healthy volunteers (mean ± SD; 11.5 ± 5 × 10(12)/ml versus 10.0 ± 4 × 10(12)/ml, respectively; P = 0.03), which correlated with the homeostasis model of insulin resistance (r = 0.53, P = 0.03). This difference was predominantly seen in MPs whose size was in the small exosomal range (<150 nm in diameter, P< 0.05). PCOS patients showed a greater percentage of annexin V(+) MPs compared with healthy volunteers (84 ± 18 versus 74 ± 24%, respectively, P = 0.05) but the cellular origin of MPs, which were predominantly platelet-derived (PCOS: 99 ± 0.9%; controls: 99 ± 2.5%), did not differ. MP fatty acid concentration and composition was similar between groups but 16 miRs were differentially expressed (P < 0.05). Patients with PCOS were classified by the Rotterdam criteria, which describes a less severe metabolic phenotype than other definitions of the syndrome. Our findings may thus not be generalizable to all patients with PCOS. MicroRNA expression analysis was only undertaken in an exploratory subset of the overall study population hence, validation of our findings in a larger cohort is mandatory. Furthermore, miR levels were unaltered for the highly expressed miRs and it is unclear whether differences in the lowly expressed miRs carries pathological relevance. This study suggests that women with PCOS have an altered MP profile but further studies are needed to confirm this, to explore the mechanisms by which these alterations develop and to establish whether therapies that improve insulin sensitivity are able to reduce circulating MP concentrations. The study was funded by grants from the Wales Heart Research Institute and Mrs John Nixon Scholarship. The authors have no conflicts of interest to declare.

Procoagulant activity and cellular origin of microparticles in human amniotic fluid

Amniotic fluid contains various procoagulant factors involved in intro-vascular amniotic fluid-induced coagulopathies. During the progression of normal pregnancy, microparticles would be shed off from cells and accumulate in amniotic fluid over time. In this study, our aims were to investigate the cellular origin and procoagulant entity of these microparticles. Twenty amniotic fluid samples from healthy parturient women were collected, and the microparticles were isolated and stained with phycoerythrin-labeled antibodies to CD138, CD41a, CD144 and CD11b to identify their cellular origin. Their phosphatidylserine and tissue factor expression levels were quantified with fluorescein isothiocyanate-labeled annexin V and anti-tissue factor antibody staining. Their procoagulant activity was tested with plasma coagulation assay and factor Xase and prothrombinase assays. Phenotypic analysis showed 36.8% and 33.8% of amniotic fluid microparticles positive for CD138 and CD11b, respectively, indicating their epithelial cell or leukocyte origin. Of these microparticles, 66.3±5.9% expressed phosphatidylserine while 37.4±4.1% expressed tissue factor. In addition, amniotic fluid microparticles could significantly shorten the plasma coagulation time and increase the production of factor Xa and thrombin. Inhibition assays with annexin V and anti-tissue factor antibody confirmed the coagulation effects of amniotic fluid microparticles. The microparticles derived from epithelial and leukocytes may be a mechanism of amniotic fluid-induced coagulopathies.

Postprandial changes in the phospholipid composition of circulating microparticles are not associated with coagulation activation

IntroductionEvidence is present that the phospholipid composition of circulating cell-derived microparticles (MP) affects coagulation in vivo, and that postprandial metabolic alterations may be associated with hypercoagulable state. Our objective was to investigate whether postprandial metabolic responses affect the phospholipid composition of MP, and whether such changes are associated with coagulation activation. Materials and MethodsTwelve healthy males were studied twice and randomly received two consecutive meals or remained fasted. Blood was collected before and at 2, 4, 6 and 8h following breakfast. Plasma concentrations of prothrombin-F1+2 and thrombin-antithrombin-complexes were measured. Numbers and cellular origin of MP were determined by flowcytometry. The phospholipid composition of MP was determined by hpTLC. In vitro procoagulant activity of MP was studied by fibrin generation. ResultsDuring the meal visit, plasma glucose, triglyceride and insulin levels increased, compared to baseline and the fasting visit (all P<0.05). Postprandially, the total numbers of MP increased in time compared to the fasting visit (P<0.05). Erythrocyte-derived MP increased (6-fold) during the meal visit, but remained constant on the fasting day (P<0.001). On the meal versus fasting day circulating MP contained increased phosphatidylcholine (P<0.05) and decreased sphingomyelin (P<0.05) amounts. The amount of phosphatidylserine did not change. Concentrations of plasma F1+2 and thrombin-antithrombin were similar on both days, as was the ability of MP to generate fibrin in vitro. ConclusionAlthough numbers, cellular origin and phospholipid composition of MP alter during exposure to two consecutive meals in healthy subjects, this does not lead to changes in the coagulation activation in vivo.

Optical and non-optical methods for detection and characterization of microparticles and exosomes.

Microparticles and exosomes are cell-derived microvesicles present in body fluids that play a role in coagulation, inflammation, cellular homeostasis and survival, intercellular communication, and transport. Despite increasing scientific and clinical interest, no standard procedures are available for the isolation, detection and characterization of microparticles and exosomes, because their size is below the reach of conventional detection methods. Our objective is to give an overview of currently available and potentially applicable methods for optical and non-optical determination of the size, concentration, morphology, biochemical composition and cellular origin of microparticles and exosomes. The working principle of all methods is briefly discussed, as well as their applications and limitations based on the underlying physical parameters of the technique. For most methods, the expected size distribution for a given microvesicle population is determined. The explanations of the physical background and the outcomes of our calculations provide insights into the capabilities of each method and make a comparison possible between the discussed methods. In conclusion, several (combinations of) methods can detect clinically relevant properties of microparticles and exosomes. These methods should be further explored and validated by comparing measurement results so that accurate, reliable and fast solutions come within reach.

Elevated Procoagulant Microparticles Expressing Endothelial and Platelet Markers in Essential Thrombocythemia

Background: Most cell types, including blood - and vascular cells, produce microparticles (MPs) upon activation. Since cellular MPs are known to be elevated in thromboembolic diseases, we hypothesized a role for MPs in the pathogenesis of thrombosis in Essential Thrombocythemia (ET).Design and methods: In plasma samples from 21 ET patients and 10 healthy subjects, the levels and the cellular origin of MPs were determined by flowcytometric analysis, while the MP-associated procoagulant activity was measured by the thrombin generation assay.Results: ET patients had significantly higher numbers of circulating AnnexinV-positive MPs than controls (median 4500 vs 2500×106 events/L; p=0.039), including significantly higher number of MPs positive for the platelet marker CD61 (median 4000 vs 2400×106/L; p=0.043) the endothelial marker CD62E (median 875 vs 14×106/L; p=0.009), and for Tissue Factor (median 1.8 vs 0.9×106/L; p=0.036). CD62E was co-expressed with the platelet marker CD41 on MPs, suggesting a bilineal origin of such MPs, that were observed only in patients with risk factors for thrombosis. ET patients had higher plasma mature von Willebrand factor (vWF) levels (median 50 vs 35 nM, p=0.045) but similar propeptide levels (median 7 vs 5 nM, p=0,07) compared to controls, indicating chronic endothelial activation. In thrombin generation analyses, MP rich plasma from ET patients had a shorter lag time (9.7 min, 95%CI: 8.7–10.7 versus 15.9 min, 95%CI: 10.9–20.9, p=0.001) and higher peak height (215 nM, 95%CI: 189–241 versus 142 nM, 95%CI: 87–189, p=0.038) than from controls. Peak height correlated significantly with the total number of MPs (R=0.634, p<0.001).Conclusions: ET patients showed higher number of circulating MPs with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This is confirmed by an increased mature vWF level and an abnormal mature vWF/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for MPs in thrombosis in ET.

Generation of Procoagulant Microparticles in Cerebrospinal Fluid and Peripheral Blood After Traumatic Brain Injury

Traumatic brain injury (TBI) can induce cell damage. Procoagulant microparticles (MPs) are reliable markers of cell stimulation. The aim of this study was to investigate the generation of procoagulant MPs in the cerebrospinal fluid (CSF) and plasma of patients with severe TBI. CSF and plasma MPs of 16 patients with severe TBI were quantified by functional prothrombinase assay (i) on the day of the trauma, (ii) during a 10-day follow-up and compared with control samples. The cellular origin of MP was determined after capture with specific antibodies. The CSF and plasma of patients with severe TBI revealed a significantly increased generation of MP compared with control samples on the day of the trauma (CSF: 4.5 +/- 1.8 vs. 0.83 +/- 0.28 nanomolar PhtdSer equivalent; p = 0.01 and plasma 4.1 +/- 3.7 vs. 2.3 +/- 0.19 nanomolar PhtdSer equivalent; p = 0.02). Procoagulant MPs were mainly of platelet and endothelial origin in CSF. MPs decreased significantly in the CSF 10 days after TBI. In CSF, a sustained generation of procoagulant MP was evidenced in two patients presenting a poor clinical outcome. In the blood flow, elevated amounts of procoagulant MPs were detected in three patients presenting disseminated intravascular coagulopathy during the follow-up. Procoagulant MP testifying to platelet and endothelial activation are produced in the CSF and in the plasma after severe TBI. A sustained generation of procoagulant MP in the CSF could contribute to a poor clinical outcome.

Protective effects of vitamin C on endothelium damage and platelet activation during myocardial infarction in patients with sustained generation of circulating microparticles

During myocardial infarction (MI), high levels of circulating procoagulant microparticles (MP) shed from endothelial cells and platelets diffuse prothrombotic and proinflammatory potentials crucial for the coronary prognosis. In addition to conventional treatments, we evaluated whether vitamin C treatment could modify circulating levels of procoagulant MP. Upon admission, 61 patients with MI were prospectively randomized for immediate additional vitamin C treatment. Circulating MP were quantified by functional prothrombinase assay before and after 5 days of vitamin C administration (1 g day-1). The cellular origin of MP was also assessed. In vitamin C-treated patients, the reduction in platelet-derived MP was 10% higher (P = 0.01). In patients with diabetes mellitus, dyslipidemia or more than two cardiovascular risk factors, vitamin C decreased endothelial and platelet-derived MP levels by approximately 70% and 13%, respectively. This early effect on circulating platelet and endothelial-derived MP, testifies to the importance of oxidative stress during MI. Vitamin C could prove beneficial for the outcome of patients at higher thrombotic risk.

Type 1 and type 2 diabetic patients display different patterns of cellular microparticles.

The development of vasculopathies in diabetes involves multifactorial processes including pathological activation of vascular cells. Release of microparticles by activated cells has been reported in diseases associated with thrombotic risk, but few data are available in diabetes. The aim of the present work was to explore the number and the procoagulant activity of cell-derived microparticles in type 1 and 2 diabetic patients. Compared with age-matched control subjects, type 1 diabetic patients presented significantly higher numbers of platelet and endothelial microparticles (PMP and EMP), total annexin V-positive blood cell microparticles (TMP), and increased levels of TMP-associated procoagulant activity. In type 2 diabetic patients, only TMP levels were significantly higher without concomitant increase of their procoagulant activity. Interestingly, in type 1 diabetic patients, TMP procoagulant activity was correlated with HbA(1c), suggesting that procoagulant activity is associated with glucose imbalance. These results showed that a wide vesiculation process, resulting from activation or apoptosis of several cell types, occurs in diabetes. However, diabetic patients differ by the procoagulant activity and the cellular origin of microparticles. In type 1 diabetic patients, TMP-procoagulant activity could be involved in vascular complications. Moreover, its correlation with HbA(1c) reinforces the importance of an optimal glycemic control in type 1 diabetes.