Plasma Microparticles Research Articles

Protein Biomarker Discovery Using Human Blood Plasma Microparticles.

Cells shed into the extracellular space a population of membranous vesicles of plasma membrane origin called microparticles (MP). Given the fact that MP are abundantly present in body fluids including plasma, rich in cell-type or disease-specific proteins and formed in conditions of stress and injury, they have been extensively investigated as biomarkers in various diseases. With the advancement in the mass spectrometry-based proteome analysis, the knowledge of the protein composition of plasma MP (PMP) has been intensively expanded, which aids the discovery of novel diagnostic target proteins. However, the lack of standardized and accurate protocols for PMP isolation limits the implementation of PMP as biomarkers in clinical settings. Here, we describe in detail a robust protocol for PMP isolation from human blood plasma via ultracentrifugation followed by label-free quantitative proteome analysis of PMP.

Diagnostic and prognostic relevance of microparticles in peripheral and uterine blood of patients with endometrial cancer

Exosomes - microvesicles which are secreted by living cells - can be produced from different cell types and detected in various body fluids. They are the carriers of intercellular information which regulate tumor microenvironment and are considered to be involved in tumor progression and metastasis. Cancer cells can secrete more exosomes than healthy cells, and are expected to be potential tools for tumor diagnosis and treatment. In this report, we present the results of microparticle analysis in peripheral and uterine blood of patients with endometrial cancer. To the best of our knowledge, this study has been the first to report microvesicle status in peripheral and uterine blood samples. The aim of the study was to determine the amount of total (TF+), endothelial (CD144+) and monocytic (CD14+) microparticles. The counting of the selected microparticles in citrate plasma was performed using flow cytometry on the BD Canto II cytometer. We found that the total amount of microparticles in cancer patients was much higher than in healthy controls. Moreover, microparticle count in uterine blood was higher than in peripheral blood of patients with endometrial cancer. We also demonstrated that the amount of microparticles correlates with the histologic grade and clinical stage of the tumor. The most interesting finding in this work was the high level of TF, CD144 and CD14 MPs in uterine blood samples. Thus we can consider the monocyte-macrophage-derived MPs as a candidate marker of endometrial cancer and maybe very critical part of the endometrial carcinogenesis.

Microparticles Carrying Sonic Hedgehog Are Increased in Humans with Peripheral Artery Disease.

Sonic hedgehog (Shh) is a prototypical angiogenic agent with a crucial role in the regulation of angiogenesis. Experimental studies have shown that Shh is upregulated in response to ischemia. Also, Shh may be found on the surface of circulating microparticles (MPs) and MPs bearing Shh (Shh + MPs) have shown the ability to contribute to reparative neovascularization after ischemic injury in mice. The goal of this study was to test the hypothesis that, in humans with peripheral artery disease (PAD), there is increased number of circulating Shh + MPs. This was done by assessing the number of Shh + MPs in plasma of patients with PAD and control subjects without PAD. We found significantly higher number of Shh + MPs in plasma of subjects with PAD, compared to controls, while the global number of MPs—produced either by endothelial cells, platelets, leukocytes, and erythrocytes—was not different between PAD patients and controls. We also found a significant association between the number of Shh + MPs and the number of collateral vessels in the ischemic limbs of PAD patients. Interestingly, the concentration of Shh protein unbound to MPs—which was measured in MP-depleted plasma—was not different between subjects with PAD and the controls, indicating that, in the setting of PAD, the call for Shh recapitulation does not lead to secretion of protein into the blood but to binding of the protein to the membrane of MPs. These findings provide novel information on Shh signaling during ischemia in humans, with potentially important biological and clinical implications.

Extracellular Vesicles: Overview and Clinical Implications

The release of extracellular vesicles (EVs) is a phenomenon shared by all cell types as a means of communication. Characterization and classification of EVs is challenging and still a matter of debate. However, a general consensus on EVs nomenclatura has been made, on the basis of their size, formation and release mechanisms. EVs has been classified into exosomes, secreted via exocytosis from the late endosome multivesicular bodies, whereas microvesicles (MVs) bud from the plasma membrane and apoptotic bodies are released by cells undergoing apoptosis. Initially regarded as cellular debris, EVs have gained considerable interest in basic sciences and medical research, both as biomarkers and mediators of biological functions. Indeed, EVs carry regulatory molecules including lipid, proteins and different RNA species through the extracellular spaces and deliver these cargos to target cells to modify cellular activity, thereby contributing to both physiological and pathological responses. Because EVs bear markers derived from their parent cells and can be detected in most body fluids, characterization of EVs of different cellular origin is an underestimated source of biological information on cellular activation during disease evolution, and will probably serve as valuable diagnostic and prognostic biomarker in the future. However, the current methods used for the EVs isolation and analysis have several limitations and lack standardization, leading to uncertainties regarding the subtypes of EVs studied and how to interpret the data.After a rapid overview of the current knowledge on the mechanisms of formation, subcellular origin and composition of the different types of EVs, this presentation will focus on microvesicles (MVs). We will first address how their structure/function diversity determines their multifaceted biological functions in coagulation, inflammation, angiogenesis and endothelial dysfunction (ref 1, 2, 3). Secondly, we will summarize the current debate on the different methodologies available for their analysis and quantification in body fluids. A specific attention will be devoted to standardization of sample processing and MVs analysis, and also to recommendations from scientific societies for EVs translational applications as emerging biomarkers measurable in liquid biopsies (ref 4, 5, 6). Finally, focusing on cardiovascular diseases and cancer as emerging field where MVs detection have promising impact to improve patient management, we will illustrate how combination of increasing fundamental knowledge, technological progress and standardization will push MVs towards reliable biomarkers ready for the clinics.1- Ridger VC, Boulanger CM, Angelillo-Scherrer A, Badimon L, Blanc-Brude O, Bochaton-Piallat ML, Boilard E, Buzas EI, Caporali A, Dignat-George F, Evans PC, Lacroix R, Lutgens E, Ketelhuth DFJ, Nieuwland R, Toti F, Tunon J, Weber C, Hoefer IE. Microvesicles in vascular homeostasis and diseases. Position Paper of the European Society of Cardiology (ESC) Working Group on Atherosclerosis and Vascular Biology. Thromb Haemost. 2017;28;117(7):1296-1316.2- Todorova D, Simoncini S, Lacroix R, Sabatier F, Dignat-George F. Extracellular Vesicles in Angiogenesis. Circ Res. 2017;12;120(10):1658-1673.3- Lacroix R, Dubois C, Leroyer AS, Sabatier F, Dignat-George F. Revisited role of microparticles in arterial and venous thrombosis. J Thromb Haemost. 2013;11 Suppl 1:24-35.4- Coumans FAW, Brisson AR, Buzas EI, Dignat-George F, Drees EEE, El-Andaloussi S, Emanueli C, Gasecka A, Hendrix A, Hill AF, Lacroix R, Lee Y, van Leeuwen TG, Mackman N, Mäger I, Nolan JP, van der Pol E, Pegtel DM, Sahoo S, Siljander PRM, Sturk G, de Wever O, Nieuwland R. Methodological Guidelines to Study Extracellular Vesicles. Circ Res. 2017;12;120(10):1632-1648.5-Lacroix R, Judicone C, Mooberry M, Boucekine M, Key NS, Dignat-George F; The ISTH SSC Workshop. Standardization of pre-analytical variables in plasma microparticle determination: results of the International Society on Thrombosis and Haemostasis SSC Collaborative workshop. J Thromb Haemost. 2013 Apr 2.6- Cointe S, Judicone C, Robert S, Mooberry MJ, Poncelet P, Wauben M, Nieuwland R, Key NS, Dignat-George F, Lacroix R. Standardization of microparticle enumeration across different flow cytometry platforms: results of a multicenter collaborative workshop. J Thromb Haemost. 2017;15(1):187-193. DisclosuresNo relevant conflicts of interest to declare.

The Release of Monocyte-Derived Tissue Factor-Positive Microparticles Contributes to a Hypercoagulable State in Idiopathic Membranous Nephropathy.

Aim: Idiopathic membranous nephropathy (IMN) is an immune-mediated inflammatory disease characterized by a high risk of thromboembolic complications. Microparticles (MPs), a type of extracellular vesicles, have procoagulant properties, especially when they display tissue factor (TF). This study aimed to investigate whether circulating TF-positive MPs contributed to the hypercoagulable state in patients with IMN.Methods: Twenty adult IMN patients and fourteen healthy subjects were included in the study. The basic indexes of a routine biochemical examination and coagulative function were determined. The plasma levels of MPs were detected by flow cytometry, and TF activity of MPs was examined using an assay kit. The plasma levels of lipopolysaccharide (LPS) were measured by an enzyme-linked immunosorbent assay.Results: Total circulating MPs were not increased in patients with IMN compared with healthy controls. Circulating CD14+/TF+MPs were significantly increased in IMN patients, but this achieved significance was not observed in CD41+/TF+MPs between the two groups. Interestingly, the circulating TF-positive MPs were increased significantly. Plasma MPs TF assays revealed high procoagulant activity, which was positively associated with the D-dimer level in IMN. In addition, circulating LPS in IMN patients were significantly higher than those in the controls. Furthermore, after two hours' incubation with healthy whole blood, LPS enhanced the release of circulating TF-positive MPs and the TF activity of MPs.Conclusion: Increased circulating LPS may mediate the release of monocyte-derived TF-positive MPs, which further contributes to the hypercoagulable state in IMN patients. These findings provide an additional mechanism by which patients with IMN have a higher risk of thromboembolic complication.

Abstract 17039: Neutrophil Subset in Blood Contributes to Acute Oxidant Signals in the Bone Marrow, Which Regulate Innate Immune Responses After Ischemic Tissue Damage

Blood neutrophils are short-lived and have distinct subsets. Aged (senescent) neutrophils are cleared by macrophages in multiple organs including bone marrow (BM), at least, in the steady state. It is unknown, after ischemic tissue damage, how these aged-type neutrophils regulate bone marrow microenvironment and impact on downstream bone marrow responses. In a mouse model of critical limb ischemia, we found that CD62L lo aged-type neutrophils that return to the BM, measured by in vivo circulating cell-labeling, are increased (2.1-fold) after acute limb ischemia, which is corresponded with acute increase in CXCR4, a major chemokine receptor promoting BM returning of blood neutrophils (31%). Interestingly, neutrophil uptake by macrophages is reduced (by 76%) in the BM after limb ischemia, suggesting increased retention of blood-derived CD62L lo neutrophils in BM microenvironment after ischemia. We also found, in the BM, that blood-derived neutrophils have higher reactive oxygen species (ROS) than other neutrophils (61% in MFI) and that limb ischemia indeed increases ROS (34%) and oxidized lipid microparticles (61%) in BM plasma in a Nox2 NADPH oxidase-dependent manner. These suggest that increased returning and retention of blood-derived CD62L lo neutrophils in the BM may contribute to oxidant signals in the BM after limb ischemia. Indeed, using aged neutrophils generated ex vivo and adoptive transfer of these cells, we found that aged neutrophils modulate BM innate immune responses after limb ischemia through ROS—reduced activation of hematopoietic stem progenitor cells (50%); increased acute activation of myeloid progenitor cells (4%); and increased acute but inhibited later Ly6C hi inflammatory monocytes/macrophages in the ischemic tissue (72% and 45%, respectively)—resulting in improved tissue recovery from ischemic damage (2.3-fold). In summary, an aged-type neutrophil subset in blood contributes to acute oxidant signals in the BM, which coordinate innate immune responses to promote tissue recovery from ischemic damage.

A10228 Endothelial microparticles from vegf inhibitor (vegfi)-treated cancer patients mediates endothelial cell signaling and et-1 production

Objectives: Vascular endothelial growth factor receptor inhibitors (VEGFRi), used as anti-angiogenic drugs to treat cancer, causes hypertension where mechanisms are still not fully elucidated. Microparticles (MPs) are biomarkers of cell damage. MPs also act as biovectors and influence vascular cell signalling and function. We questioned whether plasma levels of endothelial MPs from cancer patients treated with VEGFi are elevated (possibly reflecting underlying vascular injury) and whether these MPs influence endothelial cell signalling. Methods: Plasma MPs were isolated from cancer patients before and after treatment with VEGFRi (sunitinib, pazopanib or sorafenib). Human aortic endothelial cells (HAEC) were stimulated with isolated MPs (10*6 MPs/mL). MPs were characterized by flow cytometry; protein and gene expression by immunoblotting and qPCR; ROS and NO production by lucigenin and immunofluorescence. Results: VEGFRi treatment increased plasma endothelial cells-derived MPs (1.5 fold; p < 0.05 vs before treatment). HAEC exposed to post-treatment MPs increased pre-pro-ET-1 mRNA (9.1 fold; p < 0.01). Post-treatment MPs also increased ROS generation (5 min: 1.2 fold, p < 0.01; 30 min: 1.6 fold, p < 0.01; 60 min: 1.8 fold, p < 0.05) and phosphorylation of the inhibitory site of eNOS (Thr495) in HAEC (1.8 fold; p < 0.05). NO levels in HAEC exposed to MPs were decreased (1.2 fold; p < 0.05), an effect blocked by an ETB antagonist BQ788. Inflammatory marker gene expression was increased only in HAEC exposed to post-treatment MPs (p < 0.01). Conclusion: In conclusion, our data demonstrate that VEGFi treatment causes an increase in circulating MPs and that play a role in endothelial cell signalling and ET-1 production. These molecular processes may contribute to vascular dysfunction and hypertension in VEGFi-treated cancer patients.

Mononuclear-cell-derived microparticles attenuate endothelial inflammation by transfer of miR-142-3p in a CD39 dependent manner.

Plasma microparticles (MP) bear functional active ectonucleotidases of the CD39 family with implications in vascular inflammation. MP appear to be able to fuse with cells and transfer genetic information. Here, we tested whether levels of different immunomodulatory microRNAs (miRs) in plasma MP are modulated by CD39 after experimental hepatectomy. We further investigated whether horizontal transfer of miR-142-3p between mononuclear (MNC) and endothelial cells via MP is regulated by purinergic signaling. Partial hepatectomy was performed in C57BL/6 wild type and Cd39 null mice. MP were collected via ultracentrifugation. MNC were stimulated with nucleotides and nucleosides, in vitro, and tested for miR-142-3p levels. Fusion of MNC-derived MP and endothelial cells with subsequent transfer of miR-142-3p was imaged by flow cytometry and confocal microscopy. Endothelial inflammation and apoptosis were quantified after transfection with miR-142-3p. Significantly lower miR-142-3p levels were observed in plasma MP of Cd39 null mice after partial hepatectomy, when compared to C57BL/6 wild types (p < 0.05). In contrast to extracellular nucleotides, anti-inflammatory adenosine significantly increased miR-142-3p levels in MNC-derived MP, in vitro (p < 0.05). MNC-derived MP are able to transfer miR-142-3p to endothelial cells by fusion. Transfection of endothelial cells with miR-142-3p decreased TNF-α levels (p < 0.05) and endothelial apoptosis (p < 0.05). MiR-142-3p levels in MNC-derived MP are modulated by nucleoside signaling and might reflect compensatory responses in vascular inflammation. Our data suggest the transfer of genetic information via shed MP as a putative mechanism of intercellular communication-with implications in organ regeneration.

Type 2 diabetes and older age contribute to elevated plasma microparticle concentrations independent of chronic stroke.

What is the central question of this study? What is the effect of chronic stroke on circulating microparticle populations, accounting for potential effects of age and type 2 diabetes? What is the main finding and its importance? Elevated concentrations of CD31+ /CD42b- and CD62E+ microparticles appear to be driven by type 2 diabetes but not chronic stroke and are associated with fasting glucose and triglyceride levels. Older age results in elevations in CD62E+ and CD34+ microparticle concentrations. These microparticles have been proposed as potential targets for diagnosing, treating and identifying the clinical progression and complications of type 2 diabetes. The elevated circulating concentration of endothelial microparticles (MPs) may provide an index of the extent and nature of cellular damage in chronic stroke. The purpose of this study was to determine the circulating concentrations of CD31+ /CD42b- , CD62E+ and CD34+ MPs in chronic stroke subjects, focusing on the effects of chronic stroke by comparison with both older adults without a history of stroke but with type 2 diabetes mellitus (T2DM) and older and young healthy controls. Plasma from three groups of sedentary older (50-75years) men and women (chronic stroke, T2DM or older healthy) as well as a group of younger (18-39years) healthy controls was isolated from fasting blood, and CD31+ /CD42b- , CD62E+ and CD34+ MPs were quantified using flow cytometry (n= 17/group). Concentrations of CD31+ /CD42b- and CD62E+ MPs were higher in the T2DM group (P< 0.05), but not chronic stroke, compared to older and younger healthy adults. CD62E+ MP and CD34+ MP concentrations were elevated in the older compared to younger adults (P< 0.05 for both). Sub-analyses excluding chronic stroke subjects who were also diagnosed with diabetes [stroke (diabetes- )] revealed lower CD31+ /CD42b- (P< 0.05) and CD62E+ (P= 0.08) MPs in the stroke (diabetes- ) group compared to the T2DM group. CD31+ /CD42b- MP and CD62E+ MP concentrations were each associated with fasting glucose levels and CD31+ /CD42b- MPs also were associated with triglyceride levels. As MPs have been proposed as potential targets for diagnosing, treating and identifying the clinical progression of T2DM, our study provides further support for the use of CD31+ /CD42b- and CD62E+ MPs in the clinical progression of T2DM and associated vascular complications.

Abstract 049: Endothelial-Derived Microparticles as Biomarkers and Mediators of Endothelial Cell Injury in VEGF Inhibitor-Treated Cancer Patients: Implications in Hypertension

Vascular endothelial growth factor receptor inhibitors (VEGFRi), used as anti-angiogenic drugs to treat cancer, induce severe hypertension although the underlying molecular mechanisms are still unclear. Endothelial microparticles (MPs) are biomarkers of endothelial injury and are also functionally active since they influence downstream target cell signalling and function. We questioned whether MP status is altered in cancer patients treated with VEGFRi and whether they influence endothelial cell function associated with vascular dysfunction in hypertension. Plasma MPs were isolated from cancer patients before and after treatment with VEGFRi. Human aortic endothelial cells (HAEC) were stimulated with human plasma-isolated MPs (10 6 MPs/mL). MP characterization was assessed by flow cytometry; protein and gene expression by immunoblotting and qPCR; ROS and NO production by lucigenin and immunofluorescence. Patients treated with VEGFRi had significantly increased in endothelial cell-derived MPs (EMP) (0.19±0.02 Pre vs. 0.31±0.04 Post-treatment). HAEC exposed to post-treatment MPs increased pre-pro-ET-1 mRNA (4.10±0.97 vs. 10.57±3.54), corroborating the raise in ET-1 levels (μg/mL: 631.9±46.1 vs. 780.2±37.2) observed in HAEC stimulated with vatalanib (VEGFRi). Post-treatment MPs increased ROS generation in HAEC (100.0 vs. 5 min- 123.4±7.7, 30 min- 162.4±22.5, 60 min- 183.9±44.7), effects that were attenuated by ET A and ET B receptor blockers. VEGFRi post-treatment MPs increased phosphorylation of the inhibitory site of eNOS (Thr 495 ) (100.0 vs. 181.3±18.7) and decreased NO levels in HAEC (100.0 vs. 72.7±9.9) which was inhibited by ET B receptor blockade (eNOS: 109.8±14.9; NO: 142.1±25.9). Gene expression of proinflammatory mediators was increased in HAEC exposed to post-treatment MPs (1.0 vs. TNF-α: 5.4±1.8, MCP-1: 2.1±0.3, iNOS: 3.1±0.8, COX2: 2.4±0.4, ICAM1: 2.7±0.6), effects inhibited by BQ123 and BQ788. In conclusion, our data identify EMPs as biomarkers of VEGFi-induced endothelial injury and important mediators of ET-1-sensitive redox-regulated endothelial cell signalling. These molecular processes may play a role in vascular dysfunction associated with hypertension in VEGFRi-treated cancer patients.

Surface modification of metallic materials designed for a new generation of artificial heart valves.

The main goal of this work was to develop haemocompatibile thin film materials dedicated to novel flexible mechanical heart valves intended for pulsatile ventricle assist devices. The studies performed have led to the selection of a material for the surface modification of the metallic scaffold. Haemocompatible, biofunctional, ultra-elastic, thin carbon-based coatings were proposed. The surface was designed to eliminate thrombogenic and microbial construction by a reduction in turbulence and sufficient washing of the biofunctional-adapted surfaces, thus allowing for extended use for temporary heart support. The article presents the influence of the mechanical properties of coatings and their influence on the haemocompatibility. In this study, we investigated a simplified model of the whole blood shear stress based on a cone and plate rotational viscometer. Several indices of platelet activation were analysed, including platelet and granulocyte-platelet aggregates, platelet activation markers and platelet-derived microparticles. The shear stress induced a platelet aggregate count in the range from 2% to 30% of the CD61 positive cells. For polyurethane (PU), the average value of platelet aggregates was on the level of 7%. The analyses have demonstrated that the cytometric methods of the direct determination of platelet-derived microparticles in plasma are reproducible and reliable. Considering the generation of microparticles on the tested coatings under hydrodynamic conditions, the best properties were observed for the coating a-C:H,N. The results indicate that a-C:H-based coatings with the thickness of 110 nm do not induce an immune response and do not influence the origin of platelet microparticle formation; thus, these type of coatings are the most promising for the parts which are planned to withstand blood contact under the high value of shear stress.

Effects of donor age, donor sex, blood-component processing, and storage on cell-derived microparticle concentrations in routine blood-component preparation

BackgroundA number of factors cause increases in the number of cell-derived microparticles (MPs) in blood components. However, the overall effects of these factors on the concentration of MPs during routine blood-component preparation have not fully been elucidated. AimTo evaluate the effects of donor age, donor sex, blood-component preparation, and storage on MP concentrations. MethodsFlow cytometry was used to quantitate the number of whole blood-derived MPs. ResultsThe total MP concentration was similar in male and female donors (26,044 ± 1254 particles/μL vs. 27,696 ± 1584 particles/μL). The total MP concentration did not differ significantly among the different age groups: 18–30 years (28,730 ± 1600 particles/μL), 31–40 years (24,972 ± 5947 particles/μL), and 41–58 years (25,195 ± 1727 particles/μL). However, the total number of MPs in fresh plasma (152,110 ± 46,716 particles/μL) was significantly higher (p < 0.05) than that in unprocessed whole blood (26,752 ± 985 particles/μL), fresh packed red blood cells (PRBCs) (28,574 ± 1028 particles/μL), and platelet concentrate (PC) (33,072 ± 1858 particles/μL). Furthermore, the total numbers of MPs in stored PRBCs and fresh-frozen plasma (FFP) were significantly higher (p < 0.05) than those in fresh PRBCs and fresh plasma, respectively. ConclusionsThe study suggests that donor factors, blood-component processing and storage contribute to the MP concentration in routine blood-product preparation. The findings can improve quality control and management of blood-product manufacturing in routine transfusion laboratories.

Endothelial Microparticles and Systemic Complement Activation in Patients With Chronic Kidney Disease.

BackgroundEndothelial microparticles are associated with chronic kidney disease (CKD) and complement activation. We hypothesized that the complement pathway is activated in patients with CKD via endothelial microparticles and that complement activation correlates with endothelial dysfunction in CKD.Methods and ResultsWe analyzed complement data of 30 healthy subjects, 30 patients with stage III/IV CKD, and 30 renal transplant recipients with stage III/IV CKD, evaluating the potential correlation of complement fragments with brachial artery flow–mediated dilation, Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and urinary albumin/creatinine ratio. Endothelial microparticles were characterized via proteomic analysis and compared between study groups. Complement fragment Ba was significantly increased in CKD and post–kidney transplant CKD. Plasma Ba levels correlated significantly with lower brachial artery flow–mediated dilation, lower Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and higher urinary albumin/creatinine ratio. Factor D levels were significantly higher in the plasma microparticles of patients with CKD versus healthy controls. Plasma microparticles isolated from patients with CKD and containing factor D activated the alternative pathway in vitro.ConclusionThe alternative complement pathway is activated in CKD and correlates with endothelial dysfunction and markers of CKD. Future studies are needed to evaluate whether endothelial microparticles with increased factor D play a pathologic role in CKD‐associated vascular disease.Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02230202.

High-Mobility Group Box 1 From Hypoxic Trophoblasts Promotes Endothelial Microparticle Production and Thrombophilia in Preeclampsia.

Thrombophilia is a major complication in preeclampsia, a disease associated with placental hypoxia and trophoblast inflammation. Preeclampsia women are known to have increased circulating microparticles that are procoagulant, but the underlying mechanisms remain unclear. In this study, we sought to understand the mechanism connecting placental hypoxia, circulating microparticles, and thrombophilia. We analyzed protein markers on plasma microparticles from preeclampsia women and found that the increased circulating microparticles were mostly from endothelial cells. In proteomic studies, we identified HMGB1 (high-mobility group box 1), a proinflammatory protein, as a key factor from hypoxic trophoblasts in stimulating microparticle production in human umbilical vein endothelial cells. Immunodepletion or inhibition of HMGB1 in the conditioned medium from hypoxic human trophoblasts abolished the endothelial microparticle-stimulating activity. Conversely, recombinant HMGB1 stimulated microparticle production in cultured human umbilical vein endothelial cells. The microparticles from recombinant HMGB1-stimulated human umbilical vein endothelial cells promoted blood coagulation and neutrophil activation in vitro. Injection of recombinant HMGB1 in pregnant mice increased plasma endothelial microparticles and promoted blood coagulation. In preeclampsia women, elevated placental HMGB1 expression was detected and high levels of plasma HMGB1 correlated with increased plasma endothelial microparticles. Our results indicate that placental hypoxia-induced HMGB1 expression and release from trophoblasts are important mechanism underlying increased circulating endothelial microparticles and thrombophilia in preeclampsia.

Brain Microvascular Endothelial Derived Microparticles as a Non‐Invasive Diagnostic Tool for Multiple Sclerosis

BackgroundMultiple sclerosis (MS), an autoimmune‐neurodegenerative disease, exhibits features of neurovascular inflammation including endothelial activation and shedding of microparticles (MPs). While the brain lacks ‘classical’ lymphatics, we previously reported expression of venous‐lymphatic endothelial markers (LYVE‐1, podoplanin, prox‐1, VEGFR‐3, VEGF‐C, VEGF‐D) in both brain tissue and sera, with altered expression of these proteins in MS. Based on differential expression of these markers in brain tissue, we propose that these markers may originate within brain microvascular endothelium and undergo changes in disease which can be analyzed diagnostically.ObjectivesTo validate the neurolymphatic biomarker matrix as a novel, non‐invasive and cost‐effective tool for diagnosing MS and discriminating MS subtypes.MethodsNeurolymphatic biomarker expression was determined in human cerebral microvascular endothelial cells (hCMECs) following 24–48 hours of TNF‐α (20 ng/ml) exposure +/− IFN‐γ (1000 U/ml) using western blotting. MPs from these cells were visualized using scanning electron microscopy. Human sera were analyzed by flow cytometry, western and dot blotting to characterize relative partitioning of neurolymphatic biomarkers in MP.ResultshCMECs released MPs that contain lymphatic and junctional markers as well as caveolin‐1. Plasma MPs were seen to liberate soluble (non‐particulate) neurolymphatic biomarkers upon storage, indicating that serum represents a more stable MP‐derived pool of these markers. MP flow analysis of intracellular actin revealed that permeabilization destabilizes phosphatidylserine‐Annexin V interaction which is used to designate MP identity, suggesting that flow cytometry of MPs may require more judicious interpretation. All neurolymphatic markers so far tested were able to distinguish MS from healthy control serum samples; FOXC2 expression can further distinguish subtypes of MS: RRMS from SPMS.ConclusionLymphatic biomarkers in plasma and sera are expressed by brain microvascular endothelial cells and exhibit changes in expression in response to inflammatory stimuli (cytokines). Such alterations parallel transfer of these markers into caveolin‐1 ‐enriched microparticles, which represent a circulating ‘snapshot’ of the vascular surface that can be analyzed by several immune approaches, particularly following storage‐dependent solubilization of these biomarkers.Support or Funding InformationSupported by the Annette Funicello Research Fund for Neurological Diseases and the Malcolm Feist Predoctoral Fellowship from Center for Cardiovascular Diseases and Sciences, LSU Health Sciences Center‐ShreveportThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Microparticles in sickle cell disease.

Several pathophysiological pathways in sickle cell disease (SCD), the most prevalent hemoglobinopathy worldwide, result in activation of circulating blood cells and the release of submicron vesicles, so-called microparticles (MPs). MPs are candidate biomarkers in vascular disease that exhibit functional biological properties. Compared to healthy individuals, higher level of plasma MPs, mostly derived from platelets and red blood cells (RBC), has been repeatedly observed in SCD patients in their steady-state condition. In contrast, conflicting results have been obtained on the impact of SCD complications and hydroxyurea treatment on circulating MP concentrations, largely due to non-standardized pre- and analytical procedures. Several factors responsible for the increased release of MPs by RBC have been identified in SCD such as sickling/unsickling, oxidative stress and abnormal activity of RBC acid sphingomyelinase. Besides their well-known pro-coagulant effect, sickle RBC-derived MPs produced ex vivo can induce ROS production by endothelial cells and promote a pro-inflammatory and pro-adhesive phenotype that may lead to renal occlusion in SCD mice. However, the functional properties of circulating MPs in human sickle cell disease remain to be studied and fully characterized.

Meal intake increases circulating procoagulant microparticles in patients with type 1 and type 2 diabetes mellitus

Diabetes mellitus (DM) is associated with prothrombotic alterations, and postprandial hyperglycemia is an independent risk factor for cardiovascular complications. We therefore investigated whether a standardized mixed meal alters circulating microparticles (MPs) and their procoagulant activity in DM patients.Patients with DM type 1 (T1DM, n = 11) and type 2 (T2DM; n = 9) were studied before and 90 min after a standardized meal (without premeal insulin). MPs in plasma derived from platelets (PMPs), endothelial cells (EMPs), or monocytes (MMPs) were measured by flow cytometry. MP-induced thrombin generation in plasma was assessed by a calibrated automated thrombogram.In the fasting state, MPs did not differ significantly between T1DM and T2DM. Meal intake increased the following microparticles: PMPs expressing phosphatidylserine (by 55%, on average), P-selectin (by 86%), and tissue factor (TF; by 112%); EMPs expressing E-selectin (by 96%) and MMPs expressing TF (by 164%), with no significant group differences between T1DM and T2DM. There were no increments in EMPs expressing phosphatidylserine or TF. Meal intake increased MP-induced thrombin generation similarly in T1DM and T2DM with increased endogenous thrombin potential (p = 0.02) and peak thrombin (p = 0.03) and shortened time to peak (p = 0.02). Phosphatidylserine inhibition by lactadherin completely abolished MP-induced thrombin generation, while an anti-TF antibody had no effect.In conclusion, meal intake increased several types of circulating MPs in patients with diabetes mellitus. These MPs have a procoagulant potential, which is related to phosphatidylserine expression and negatively charged MP surfaces rather than to TF.

Microparticle Release During Normal Cesarean Delivery

Coagulation increases during pregnancy and peaks during parturition. We hypothesized that an increase in microparticle (MP) levels in plasma occurs around the time of placental separation and subsides over several hours. We performed a prospective observational pilot study to investigate plasma MP levels in healthy parturients immediately before and after cesarean delivery. The primary outcome was MP levels at postdelivery time points compared to baseline levels. Samples underwent flow cytometry and staining to determine MP levels. Placental-derived MPs were further characterized for the presence of procoagulant proteins. Placental-derived MPs increased immediately after delivery before returning to baseline in healthy parturients.

Pathophysiology of Trousseau’s syndrome

SummaryClinically relevant clotting abnormalities in cancer patients are referred to as Trousseau’s syndrome. While thrombotic complications such as venous thromboembolism are most frequent in every day’s practice, cancer patients may also experience severe bleeding symptoms due to complex systemic coagulopathies, including disseminated intravascular coagulation, haemolytic thrombotic microangiopathy, and hyperfibrinolysis. The pathophysiology of Trousseau’s syndrome involves all aspects of Virchow’s triad, but previous basic research has mainly focused on the cellular and molecular mechanisms underlying blood hypercoagulability in solid cancers and haematological malignancies. In this regard, over-expression of tissue factor (TF), the principal initiator of the extrinsic coagulation pathway, by primary tumour cells and increased shedding of TF-bearing plasma microparticles are critical to both thrombus formation and cancer progression. However, novel findings on intrinsic contact activation in vivo, such as the release of polyphosphates or DNA by activated platelets and neutrophils, respectively, have pointed to additional pathways in the complex pathophysiology of Trousseau’s syndrome.

Relevance of elevated microparticles in peripheral blood and synovial fluid of patients with rheumatoid arthritis

Introduction: Microparticles (MPs) are submicron sized heterogeneous membrane-bound vesicles released from cells undergoing cell activation or apoptosis. Elevated platelet-derived MPs (PMPs) are reported in rheumatic diseases. We profiled Annexin-V+ MPs and CD61+ MPs in plasma and synovial fluid of patients with rheumatoid arthritis (RA) and their clinical correlates. Methods: Twenty-three newly diagnosed disease-modifying anti-rheumatic drug naive patients with RA, 17 with osteoarthritis (OA) and 22 healthy controls (HC) were enrolled. Five milliliters of synovial fluid from the knee joint of patients with RA and OA was collected; 5 ml of peripheral blood was collected from patients with RA, OA, and HC. Cell-free synovial fluid and platelet poor plasma samples were stained with Annexin-V-APC and Anti-CD61-BV510 antibodies followed by flow cytometry analysis (FACSAria III) (results expressed as the mean ± standard deviation of % population). Results: Significantly greater levels of synovial fluid MPs (Annexin-V+ MPs) were observed in RA versus OA (P Conclusions: Higher levels of Annexin-V+ MPs in both plasma and synovial fluid of RA suggest a role for MPs in the pathogenesis of RA. Elevated plasma PMPs (Annexin V+ CD61+ MPs) in RA may suggest their role in systemic involvement. Furthermore, increased levels of MPs other than those of platelet origin (Annexin V+ CD61− MPs) indicate the necessity to study the MPs from other cell lineages.