Circulating microparticles may influence early carotid artery remodeling.

OBJECTIVES: To test associations of circulating microparticles with large artery remodeling before atherosclerosis is detectable. METHODS: In 232 untreated symptom-free individuals, we measured microparticles of various cellular origins (platelet, endothelial and leukocyte) by specific anti-GPIb (glycoprotein Ib), anti-cluster of differentiation (CD) 105 and anti-CD11a antibodies, and common carotid artery intima-media thickness (IMT), internal and external diameters by ultrasound. RESULTS: Except for CD105 microparticles with IMT to lumen ratio (IMT/D, P < 0.05), microparticles correlated with no carotid dimensions. Significant interactions existed between each microparticle type and IMT on internal and external diameters (GPIb, P < 0.01; CD105 and CD11a microparticles P < 0.001) consisting of lower trend in increased diameter with increasing IMT in individuals with high than in those with low microparticle level (according to the median) of each origin. As a result, individuals within the third IMT tertile had lower internal diameter in the presence of high than in the presence of low level of GPIb, CD105 or CD11a microparticles (P = 0.001, <0.05 or 0.01, respectively), and lower external diameter in the presence of high than in the presence of low level of GPIb and CD11a microparticles (P = 0.001 and 0.01). Also, individuals within third IMT tertile exhibited positive correlations of IMT with CD105 and CD11a microparticles (P < 0.05), negative correlations of internal diameter with GPIb (P < 0.05), CD105 (P < 0.05) and CD11a microparticles (P < 0.01) and of external diameter with GPIb and CD11a microparticles (P < 0.05), and positive correlations of IMT/D with CD105 and CD11a microparticles (P < 0.001). CONCLUSION: Increased levels of leukocyte and endothelial-derived microparticles are associated with carotid inward remodeling in individuals with the greatest IMT before atherosclerosis is detectable.

Generation of procoagulant microparticles in cerebrospinal fluid and peripheral blood after traumatic brain injury.

BACKGROUND: 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. MATERIAL: 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. RESULTS: 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. CONCLUSION: 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.

Levels of circulating procoagulant microparticles in nonvalvular atrial fibrillation.

Circulating procoagulant microparticles (MPs) arising from cell activation or fragmentation during apoptosis retain procoagulant properties and are increased in severe thrombotic states. We investigated whether circulating procoagulant MP levels would be increased in nonvalvular atrial fibrillation (AF). Using a hospital case-control study design, circulating procoagulant MP levels were measured in 45 patients with permanent and/or persistent AF who were not receiving anticoagulant therapy and 90 age-matched control subjects (45 with cardiovascular risk factors and 45 without). Annexin V-positive MP levels (expressed as nanomoles per liter of phosphatidylserine equivalent) were higher in patients with AF (median 9.3, interquartile range 6.8 to 17.3 nmol/L) than in control subjects with cardiovascular risk factors (median 4.9, interquartile range 3.7 to 8.4 nmol/L) and control subjects without cardiovascular risk factors (median 3.2, interquantile range 2.3 to 4.6 nmol/L; p<0.001). Platelet-derived MPs (captured with antiglycoprotein Ib) and endothelial-derived MPs (captured with anti-CD31) were similar in patients with AF and control subjects with cardiovascular risk factors but were significantly higher than in control subjects without cardiovascular risk factors. On multiple regression analysis, the presence of AF was a strong predictor of annexin V-positive MP level (p<.001). In conclusion, circulating procoagulant MPs are increased in persistent and/or permanent AF and might reflect a hypercoagulable state that could contribute to atrial thrombosis and thromboembolism.

Circulating leukocyte-derived microparticles predict subclinical atherosclerosis burden in asymptomatic subjects.

OBJECTIVE: To clarify circulating microparticles (MP) relationships with preclinical atherosclerosis. METHODS AND RESULTS: In 216 subjects without cardiovascular disease, we assessed: (1) annexin V-positive, platelet-derived, endothelium-derived and leukocyte-derived circulating MP by capture on annexin V, anti-GPIb, anti-CD105, and anti-CD11a antibody-coated wells, respectively; (2) Framingham risk, metabolic syndrome, and low-grade inflammation by risk factors measurement including hsCRP; and (3) subclinical atherosclerosis by ultrasound examination of carotid, abdominal aorta, and femoral arteries. Number of sites with plaque ranged from 0 to 3 and plaque burden was classified into 0 to 1 or 2 to 3 sites disease. Leukocyte-derived MP level was higher in the presence than in the absence of moderate to high Framingham risk (P<0.05), metabolic syndrome (P<0.01), high C-reactive protein (CRP) (P<0.05), or 2- to 3-sites disease (P<0.01), and correlated positively with number of metabolic syndrome components (P<0.001), tertiles of fibrinogen (P<0.001), and number of diseased sites (P<0.01). In multivariate analysis, 2- to 3-sites disease was independently associated with leukocyte-derived MP level (P<0.05), Framingham risk (P<0.001), and metabolic syndrome (P<0.01). None of the other MP types correlated with risk markers or atherosclerosis. CONCLUSIONS: Leukocyte-derived MP, identified by affinity for CD11a, are increased in subjects with ultrasound evidence of subclinical atherosclerosis, unveiling new directions for atherosclerosis research.

Procoagulant microparticles: disrupting the vascular homeostasis equation?

Apoptosis and vascular cell activation are main contributors to the release of procoagulant microparticles (MPs), deleterious partners in atherothrombosis. Elevated levels of circulating platelet, monocyte, or endothelial-derived MPs are associated with most of the cardiovascular risk factors and appear indicative of poor clinical outcome. In addition to being a valuable hallmark of vascular cell damage, MPs are at the crossroad of atherothrombosis processes by exerting direct effects on vascular or blood cells. Under pathological circumstances, circulating MPs would support cellular cross-talk leading to vascular inflammation and tissue remodeling, endothelial dysfunction, leukocyte adhesion, and stimulation. Exposed membrane phosphatidylserine and functional tissue factor (TF) are 2 procoagulant entities conveyed by circulating MPs. At sites of vascular injury, P-selectin exposure by activated endothelial cells or platelets leads to the rapid recruitment of MPs bearing the P-selectin glycoprotein ligand-1 and blood-borne TF, thereby triggering coagulation. Within the atherosclerotic plaque, sequestered MPs constitute the main reservoir of TF activity, promoting coagulation after plaque erosion or rupture. Lesion-bound MPs, eventually harboring proteolytic and angiogenic effectors are additional actors in plaque vulnerability. Pharmacological strategies aimed at modulating the release of procoagulant MPs appear a promising therapeutic approach of both thrombotic processes and bleeding disorders.

Proteomic analysis of malignant lymphocyte membrane microparticles using double ionization coverage optimization.

Shed membrane microparticles (MPs) are microvesicles generated from the plasma membrane when cells are submitted to stress conditions. Although MPs reflect the cell state (at least in vitro), little is known on their protein composition. We describe the first set of experiments aiming to characterize the MP proteome. Two ways of triggering MP formation from a T-lymphocytic cell line were analyzed using a 1-D gel approach coupled with LC-MS/MS and the results were compared with those obtained from a classic membrane preparation. In total, 390 proteins were identified in MPs, among which 34% were localized to the plasma membrane. The MPs revealed a broad representation of plasma membrane proteins including 17 hematopoietic clusters of differentiation. This approach was successfully applied to one human chronic B-cell lymphoid malignancy. In all, 413 proteins were identified, including 117 membrane proteins, many of them being pathology associated. The sequence coverage in identified proteins was improved combining both nano-LC-MS/MS and MALDI-MS data. The suppression effect, observed on very complex peptide mixtures, was remediated by chromatographic fractionation. MPs may represent a new tool for studying plasma membrane proteins, displaying the advantages of reproducibility, minimal organelle contamination, and being potentially applicable to most cell types.

Circulating microparticles are elevated in haemophiliacs and non-haemophilic individuals aged <18 years.

Platelet and cell stimulation lead to plasma membrane remodelling, resulting in phosphatidylserine (PS) externalisation in the outer leaflet of the membrane, associated with the shedding of PS-rich microparticles (MPs), and contributes to thrombin generation by promoting the assembly of coagulation enzyme complexes. A previous study assessed MP levels in haemophiliacs after haemostatic treatment. To further investigate in vivo membrane remodelling, MP levels and characteristics were studied in 79 haemophiliacs and in 62 non-haemophiliacs. Both groups showed heterogeneity in MP levels, with higher values in individuals <18 years. This finding should be considered when studying MP levels in individuals <18 years.

Upregulation of proinflammatory proteins through NF-kappaB pathway by shed membrane microparticles results in vascular hyporeactivity.

OBJECTIVE: Microparticles are membrane vesicles with procoagulant and proinflammatory properties released during cell activation, including apoptosis. The present study was designed in dissecting the effects evoked by microparticles on vascular reactivity. METHODS AND RESULTS: Microparticles from either apoptotic T lymphocytic cells or from plasma of diabetic patients with vascular complications induced vascular hyporeactivity in response to vasoconstrictor agents in mouse aorta. Hyporeactivity was reversed by nitric oxide (NO) synthase plus cyclooxygenase-2 inhibitors, and associated with an increased production of vasodilatory products such as NO and prostacyclin. Microparticles induced an upregulation of proinflammatory protein expressions, inducible NO-synthase and cyclooxygenase-2, mainly in the medial layer of the vessels as evidenced by immunochemical staining. In addition, microparticles evoke NF-kappaB activation probably through the interaction with the Fas/Fas Ligand pathway. Finally, in vivo treatment of mice with lymphocyte-derived MPs induces vascular hyporeactivity, which was reversed by the combination of NO and cyclooxygenase-2 inhibitors. CONCLUSIONS: These data provide a rationale to explain the paracrine role of microparticles as vectors of transcellular exchange of message in promoting vascular dysfunction during inflammatory diseases.

Circulating secretory phospholipase A2 activity predicts recurrent events in patients with severe acute coronary syndromes.

OBJECTIVES: The purpose of this study was to determine the prognostic value of circulating secretory phospholipase A2 (sPLA2) activity in patients with acute coronary syndromes (ACS). BACKGROUND: The plasma level of type IIA sPLA2 is a risk factor for coronary artery disease (CAD) and is associated with adverse outcomes in patients with stable CAD. The prognostic impact of sPLA2 in patients with ACS is unknown. METHODS: Secretory phospholipase A2 antigen levels and activity were measured in plasma samples of 446 patients with ACS, obtained at the time of enrollment. RESULTS: Baseline sPLA2 activity was associated with the risk of death and myocardial infarction (MI). The unadjusted rate of death and MI increased in a stepwise fashion with increasing tertiles of sPLA2 activity (p < 0.0001). The association remained significant in the subgroup of patients who had MI with ST-segment elevation (p = 0.014) and the subgroup of patients who had unstable angina or non-ST-segment elevation MI (p < 0.002). After adjustment for clinical and biological variables, the hazard ratios for the combined end point of death or MI in the third tertile of sPLA2 compared with the first and second tertiles was 3.08 (95% confidence interval, 1.37 to 6.91, p = 0.006). CONCLUSIONS: A single measurement of plasma sPLA2 activity at the time of enrollment provides strong independent information to predict recurrent events in patients with ACS.

Circulating cell-derived microparticles in Crohn's disease.

Procoagulant membrane microparticles can be released from activated or apoptotic cells in response to various environmental stimuli. The aim of this study was to investigate the presence of microparticles in Crohn's disease and to assess their variations after infliximab therapy. We compared the levels of circulating microparticles in 38 patients with Crohn's disease, 16 patients with ulcerative colitis, 7 patients with infectious colitis, and 17 control subjects. The evolution of microparticle levels was assessed after infliximab therapy in 13 patients with Crohn's disease. Circulating microparticle levels were elevated in patients with Crohn's disease (9.31+/-0.66 nmol/L phosphatidylserine equivalent [PS Eq]) or infectious colitis (10.71+/-0.92 nmol/L PS Eq) compared to patients with ulcerative colitis (5.75+/-0.59 nmol/L PS Eq) and control subjects (4.06+/-0.37 nmol/L PS Eq) (P = 0.001). Infliximab induced a significant diminution of the amounts of circulating microparticles, from 10.33+/-1.20 to 6.45+/-0.90 nmol/L PS Eq (P = 0.002). Generation of circulating microparticles occurs in Crohn's disease; infliximab induces significant diminution. Release of microparticles could be linked to the type of inflammatory response underlying Crohn's disease.

Membrane microparticles: two sides of the coin.

Microparticles are plasma membrane-derived vesicles shed from stimulated cells, in the broad sense of the term. Their presence is interpreted by proximal or remote cells in fundamental physiological processes including intercellular communication, hemostasis, and immunity. On the other hand, variations of their number or characteristics are frequently observed in pathophysiological situations.

Fractalkine reduces N-methyl-d-aspartate-induced calcium flux and apoptosis in human neurons through extracellular signal-regulated kinase activation.

Our purpose was to investigate in human neurons the neuroprotective pathways induced by Fractalkine (FKN) against glutamate receptor-induced excitotoxicity. CX(3)CR1 and FKN are expressed constitutively in the tested human embryonic primary neurons and SK-N-SH, a human neuroblastoma cell line. Microfluorometry assay demonstrated that CX(3)CR1 was functional in 44% of primary neurons and in 70% of SK-N-SH. Fractalkine induced ERK1/2 phosphorylation within 1 min and Akt phosphorylation after 10 min, and both phosphorylation decreased after 20 min. No p38 and SAPK/JNK activation was observed after FKN treatment. Application of FKN triggered a 53% reduction of the NMDA-induced neuronal calcium influx, which was insensitive to pertussis toxin and LY294002 an inhibitor of Akt pathway, but abolished by PD98059, an ERK1/2 pathway inhibitor. Moreover, FKN significantly reduced neuronal NMDA-induced apoptosis, which was pertussis toxin insensitive and abolished in presence of PD98059 and LY294002. In conclusion, FKN protected human neurons from NMDA-mediated excitotoxicity in at least two ways with different kinetics: (i) an early ERK1/2 activation which reduced NMDA-mediated calcium flux; and (ii), a late Akt activation associated with the previously induced ERK1/2 activation.

Cellular microparticles: a disseminated storage pool of bioactive vascular effectors.

PURPOSE OF REVIEW: Microparticles (MP) or microvesicles are fragments shed from the plasma membrane of stimulated or apoptotic cells. Having long been considered inert debris reflecting cellular activation or damage, MP are now acknowledged as cellular effectors involved in cell-cell crosstalk. This review focuses on procoagulant MP circulating in the vascular compartment, their role in hemostasis and thrombosis, and possible impact in vascular functions. RECENT FINDINGS: Microparticles can be viewed as a "storage pool" by themselves, disseminating blood-borne tissue factor activity and procoagulant phospholipids. Increasing evidences of integrated loops involving dynamic exchanges and transfer events through multiple MP-cell interactions are summarized. SUMMARY: Microparticles can be considered true targets in the pharmacological control of thrombosis. Another challenging issue is to take advantage of their procoagulant potential for the management of hemophilia.

Impaired clearance of apoptotic cells promotes synergy between atherogenesis and autoimmune disease.

To clarify the link between autoimmune disease and hypercholesterolemia, we created the gld.apoE(-/-) mouse as a model of accelerated atherosclerosis. Atherosclerotic lesion area was significantly increased in gld.apoE(-/-) mice compared with apoE(-/-) mice. gld.apoE(-/-) mice also displayed increases in lymphadenopathy, splenomegaly, and autoantibodies compared with gld mice, and these effects were exacerbated by high cholesterol diet. gld.apoE(-/-) mice exhibited higher levels of apoptotic cells, yet a reduced frequency of engulfed apoptotic nuclei within macrophages. Infusion of lysophosphatidylcholine, a component of oxidized low density lipoprotein, markedly decreased apoptotic cell clearance in gld mice, indicating that hypercholesterolemia promotes autoimmune disease in this background. These data suggest that defects in apoptotic cell clearance promote synergy between atherosclerotic and autoimmune diseases.

Injection of recombinant activated factor VII can induce transient increase in circulating procoagulant microparticles.

Recombinant activated factor VII (rFVIIa) is an effective haemostatic treatment in haemophiliacs with inhibitors. In vitro, FVIIa concentrations corresponding to those obtained with therapeutic doses of rFVIIa have been shown to induce normal thrombin generation and platelet activation in the absence of factors VIII or IX. To further study the in vivo haemostatic changes induced by rFVIIa, circulating procoagulant microparticles (MP) were measured in patients treated with discontinuous injections of Novoseven. In 6 out of 15 patients, a transient peak of procoagulant MP was observed after injection, occurring 15 min to 2 h after infusion. It was composed primarily of platelet-derived MP and was of very short duration. This peak was not observed in haemophiliacs without inhibitor, who were treated with conventional replacement therapies. Our results provide further in vivo evidence that rFVIIa specifically activates platelets, either directly or as a consequence of a burst of thrombin generation that could account for its haemostatic efficacy.

Shed membrane particles from T lymphocytes impair endothelial function and regulate endothelial protein expression.

BACKGROUND: Microparticles (MPs) are membrane vesicles with procoagulant and proinflammatory properties released during cell activation. The present study was designed to dissect the effects evoked by T lymphocyte-derived MPs on vascular function. METHODS AND RESULTS: MPs were produced by treatment of the human lymphoid CEM T cell line with actinomycin D or phytohemagglutinin. Incubation of mouse aortic rings with 30 nmol/L MPs resulted in a time-dependent impairment of acetylcholine-induced relaxation of precontracted vessels, with a maximal reduction after 24 hours. MPs also impaired shear stress-induced dilatation of mouse small mesenteric arteries by affecting the nitric oxide (NO) and prostacyclin but not the endothelium-derived hyperpolarizing factor components of the response. However, neither alteration of calcium signaling in response to agonists nor reduction of cyclooxygenase-1 expression accounted for the impairment of the NO and prostacyclin components of the endothelial response. The effect of MPs was rather because of a decrease in expression of endothelial NO synthase and an overexpression of caveolin-1. Furthermore, lymphocyte-derived MPs from diabetic patients or in vivo circulating MPs from either diabetic or HIV-infected patients reduced endothelial NO synthase expression. Finally, the effects of MPs on endothelial cells were not driven through CD11a/CD18 adhesion molecules or the Fas/FasL pathway. CONCLUSIONS: MPs from T cells induce endothelial dysfunction in both conductance and resistance arteries by alteration of NO and prostacyclin pathways. MPs regulate protein expression for endothelial NO synthase and caveolin-1. These data contribute to a better understanding of the deleterious effects of enhanced circulating MPs observed in disorders with cardiovascular or immune complications.

Sustained elevated amounts of circulating procoagulant membrane microparticles and soluble GPV after acute myocardial infarction in diabetes mellitus.

During myocardial infarction (MI), platelet activation and endothelial apoptosis are responsible for the release of procoagulant membrane-derived microparticles (MP) in the blood flow. MP prothrombotic and proinflammatory properties may be crucial for coronary prognosis. Elevated amounts of circulating procoagulant MP were described in diabetes mellitus (DM), and could be of particular significance in a MI context. We evaluated the prothrombotic status of DM and non-DM (NDM) patients at days 1 and 6 after MI, by measurement of circulating procoagulant MP and soluble GPV (sGPV), the platelet glycoprotein V major fragment released upon thrombin cleavage. Variations were compared to values measured in healthy volunteers (HV). Procoagulant MP were captured onto insolubilized annexin V and quantified by prothrombinase assay. Their cellular origin was assessed. With respect to HV, the levels of procoagulant MP detected at D1 and D6 were elevated in DM and NDM, MP being significantly higher in DM vs. NDM. The high amounts of platelet-derived MP and the correlation between procoagulant MP and sGPV, testify to the central role of thrombin-activated platelets during MI in both DM and NDM subsets. The release of platelet and endothelial cell-derived MP persisted at D6 and was more important in DM, the associated prothrombotic risk being also reflected by higher levels of sGPV. The endothelial damage revealed by endothelial-derived MP was twice that observed in NDM patients. In DM patients presenting cardio-vascular events at 6 month follow-up, MP levels were significantly higher at D1 after MI than in those without complication (24.9 +/- 4.8 vs. 12.3 +/- 2.7 nM PhtdSer, p = 0.02), suggesting a prognostic potential for MP.

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.

Possible pathogenic role of muscle cell dysfunction in motor neuron death in spinal muscular atrophy.

We have previously shown that myofibers formed by fusion of muscle satellite cells from spinal muscular atrophy (SMA) I or II undergo degeneration 1 to 3 weeks after innervation by rat embryonic spinal cord explants, whereas normal myofibers survive for several months. In the "muscle component" of the coculture, the only cells responsible for the degeneration are the SMA muscle satellite cells. Moreover, SMA muscle satellite cells do not fuse as rapidly as do normal muscle satellite cells. To determine whether death of muscle cells precedes that of motor neurons, we studied the origin and kinetics of release of apoptotic microparticles. In SMA cocultures, motor neuron apoptosis occurred before myofiber degeneration becomes visible, indicating that SMA myofibers were unable to sustain survival of motor neurons. In normal cocultures, motor neuron apoptosis occurred 4 days after innervation. However, it did not continue beyond 2 days. These results strengthen the hypothesis that SMA is due to a defect in neurotrophic muscle cell function.