Release Of Microparticles Research Articles

Enhanced Procoagulant Activity on Blood Cells after Acute Ischemic Stroke.

The role of phosphatidylserine (PS)-mediated procoagulant activity (PCA) in stroke remains unclear. To ascertain this role, early dynamic evolution of PS exposure on blood cells and released microparticles (MPs) and the corresponding PCA were evaluated in patients with acute ischemic stroke (AIS). Flow cytometry analyses revealed that initial levels of PS exposure on erythrocyte, platelet, and leukocyte were 2.40-, 1.36-, and 1.38-fold higher, respectively, in AIS than the risk factor-matched (RF) controls. Concomitantly, total PS+ MPs were increased in AIS (1949±483/μl) compared with the RF group (1674±387/μl; P=0.019) and healthy controls (1052±179/μl; P<0.001). Specifically, PS+ erythrocytes gradually increased within 1week. PS+ platelets and MPs peaked at 24h and declined at 7days, while PS+ leukocytes were markedly elevated at 24h. Further, PS exposure on blood cells and MPs in stroke resulted in shortened clotting time with an accompanying increase in FXa and thrombin formation significantly. Treatment with lactadherin, a PS antagonist, delayed the coagulation time by approximately 20% and blocked the generation of FXa and thrombin by about 50%. Furthermore, initial counts of PS+ platelets and platelet MPs significantly correlated with stroke severity. Thrombin generation promoted by platelets and MPs at 12h was significantly higher in patients with cardioembolism than in patients without. The thrombophilic susceptibility of AIS patients can be partly ascribed to PS exposure on blood cells and the release of MPs. Our studies identify PS exposure as a potentially novel therapeutic target in the treatment of AIS.

Recombinant tissue plasminogen activator enhances microparticle release from mouse brain-derived endothelial cells through plasmin

Thrombolysis with recombinant tissue plasminogen activator (rt-PA) is currently the only approved pharmacological strategy for acute ischemic stroke. However, rt-PA exhibits vascular toxicity mainly due to endothelial damage. To investigate the mechanisms underlying rt-PA-induced endothelial alterations, we assessed the role of rt-PA in the generation of endothelial microparticles (EMPs), emerging biological markers and effectors of endothelial dysfunction.The mouse brain-derived endothelial cell line bEnd.3 was used. Cells were treated with rt-PA at 20, 40 or 80μg/ml for 15 or 24h, and EMPs were quantified in the culture media using Annexin-V staining coupled with flow cytometry. Rt-PA enhanced EMP release from bEnd.3 cells with a maximal increase at the 40μg/ml dose for 24h (+78% compared to controls). Using tranexamic acid and aprotinin we demonstrated that plasmin is responsible for rt-PA-induced EMP release. The p38 MAPK inhibitor SB203580 and the poly(ADP-ribose)polymerase (PARP) inhibitor PJ34 also reduced rt-PA-induced EMP production, suggesting that p38 MAPK and PARP are downstream intracellular effectors of rt-PA/plasmin. Rt-PA also altered through plasmin the morphology and the confluence of bEnd.3 cells. By contrast, these changes did not implicate p38 MAPK and PARP.This study demonstrates that rt-PA induces the production of microparticles by cerebral endothelial cells, through plasmin, p38 MAPK and PARP pathways. Determining the phenotype of these EMPs to clarify their role on the endothelium in ischemic conditions could thus be of particular interest.

PP.16.24] EFFECTS OF ANTIPHOSPHOLIPID ANTIBODIES ON VASCULAR SMOOTH MUSCLE CELLS

Objective: Activation of platelets, monocytes and endothelial cells antiphospholipid antibodies (aPL) has been proposed as one of the pathogenic mechanisms contributing to the antiphospholipid syndrome (APS). We determined effects of aPL on characteristic functions of human vascular smooth muscle cells (hVSMCs). Design and method: Total IgG were purified from sera of 10 patients with APS by chromatography on protein G. Thrombin generation was monitored using calibrated automated thrombography (CAT) at the surface of adherent cultured normal hVSMCs incubated with IgG (250 μg/ml) from APS patients or healthy controls, and with or without activated protein C (APC). Proliferation of hVSMCs in response to stimulation by IgG from patients or controls was studied in medium without fetal bovine serum (FBS). Negatively-charged phospholipids were quantified in the cell supernatant. hVSMC microparticles (MPs) release was measured by flow cytometry in medium without FBS. Results: Thrombin generation was markedly increased after incubation with IgG from all patients irrespective of the aPL concentrations in sera compared to control IgG or to medium without IgG. The increase ranged from 2-fold to 4-fold for triple positive patients (anti-beta2GPI antibodies, anticardiolipin and lupus anticoagulant). In addition, IgG from the majority of APS patients induced a resistance to APC. For all patients, procoagulant activity increase without FBS in parallel with increased apoptosis (75%) compared with unstimulated cells or cells stimulated with IgG control. IgG of all patients induced significant increase of MPs released from hVSMCs (from 200% to 300%). Conclusions: The results show the activating effects of aPL on hVSMCs: thrombin generation increased and this hypercoagulability depends on aPL antibody profiles. The aPL-induced release of MPs and apoptotic cells induction may represent crucial mechanisms driving increased thrombin generation.

Effect of endothelin-1 and endothelin receptor blockade on the release of microparticles.

Increased levels of endothelial cell microparticles (EMP) are known to reflect endothelial dysfunction (ED). In diabetes mellitus type 2 (T2DM), the expression of endothelin (ET)-1 is increased. As treatment with an ET-1 antagonist significantly inhibited atherosclerosis in animal models, we sought to investigate whether treatment with ET-1 antagonists affects EMP levels in vitro and in vivo in patients with T2DM. In vitro study: Human umbilical vein endothelial cells (HUVEC) were stimulated with ET-1 alone and ET-1 in combination with a dual ET-A and ET-B endothelin receptor blocker. In vivo study: Patients with T2DM were randomized to treatment with the ET receptor antagonist bosentan or placebo. After 4 weeks, the patients were re-examined and blood samples were obtained. EMP counts in supernatants and plasma samples were determined using flow cytometry. In vitro study: In supernatants of ET-1-stimulated HUVECs, the increased release of EMP was reduced significantly by co-incubation with an ET-1 receptor antagonist (e.g. CD31+/CD42b-EMP decreased from 37·1% ± 2·8 to 31·5% ± 2·8 SEM, P = 0·0078). In vivo study: No changes in EMP levels in blood samples of patients with T2DM were found after 4 weeks of bosentan treatment (n = 36, P = ns). Our in vitro results suggest that ET-1 stimulates the release of EMP from HUVECs via a receptor-dependent mechanism. Co-incubation with an endothelin receptor blocker abolished ET-1-dependent EMP release. However, treatment with bosentan for 4 weeks failed to alter EMP levels in patients with T2DM. Other factors seem to have influenced EMP release in patients with T2DM independent of ET-1 receptor-mediated mechanisms.

Plasma levels of endothelial and B-cell-derived microparticles are restored by fingolimod treatment in multiple sclerosis patients

Background: No molecular marker can monitor disease progression and treatment efficacy in multiple sclerosis (MS). Circulating microparticles represent a potential snapshot of disease activity at the blood brain barrier. Objectives and methods: To profile plasma microparticles by flow cytometry in MS and determine how fingolimod could impact endothelial microparticles production. Results: In non-treated MS patients compared to healthy and fingolimod-treated patients, endothelial microparticles were higher, while B-cell-microparticle numbers were lower. Fingolimod dramatically reduced tumour necrosis factor (TNF)-induced endothelial microparticle release in vitro. Conclusion: Fingolimod restored dysregulated endothelial and B-cell-microparticle numbers, which could serve as a biomarker in MS.

37 1,25(OH)2D3 suppresses oxidative stress – Induced microparticle release by placental trophoblasts

Introduction Emerging evidence showed that microparticles (MPs) derived from placental trophoblasts (TC) contribute to maternal vascular dysfunction and promote endothelial inflammatory response in preeclampsia (PE). Vitamin D insufficiency has been linked to placental dysfunction in pregnancy disorders such as PE and IUGR. Objective To determine: (1) if TC from PE placentas release more MPs than TC from normal placentas; (2) if vitamin D could suppress oxidative stress-induced MP release by placental TC; and (3) potential mechanism of vitamin D protective effects on placental TCs. Methods TC were isolated from normal and PE placentas by trypsin digestion and purified with Percoll gradient centrifugation. Freshly isolated TC (5×06cells/well) were seeded into 6 well plates and cultured with DMEM containing 5% FBS and antibiotics for 72h. For the oxidative stress experiment, cobalt chloride (CoCl 2 , a hypoxic mimicking agent) was used to induce TC oxidative stress and 1,25(OH)2D3 was used as bioactive vitamin D. MPs were isolated from culture supernatants by a two-step centrifugation procedure (420g for 20min and then 20,000g for 60min) and quantified by annexin V positive staining measured by flow cytometry with TruCount beads to determine absolute counts of MP for each sample. Data are expressed as annexin V positive MP/μg cell protein/well. Total cellular protein and MP protein were also collected and protein expression for caveolin-1, eNOS, pro-caspase-3, cleaved caspase-3, and ROCK1 were determined by Western blot. Data were analyzed by paired or unpaired t-test or ANOVA with Newman-Keuls test as a post hoc test. A p level Results TC isolated from PE placentas ( n =6) released significantly more MP than cells isolated from normal placentas ( n =6), 295.20±47.67 vs. 117.20±18.02/μg protein/well, p 2 compared to control TC, p 2 was significantly reduced in TC treated with 1,25(OH)2D3+CoCl 2 , p 2 . This phenomenon could be reversed when cells were treated with 1,25(OH)2D3. Moreover, reduced pro-caspase-3 and ROCK1 expression in TC induced by CoCl 2 could also be attenuated in cells treated with1,25(OH)2D3. Conclusions MP release was significantly increased in TC from PE placenta. 1,25(OH)2D3 suppresses oxidative stress-induced MP release by placental TC. Caveolin-1 is an essential structural component of caveolae and eNOS is associated with plasma membrane caveolae. Therefore, reduced caveolin-1 and increased eNOS expression together with reduced MP release in TC treated with 1,25(OH)2D3 demonstrate that vitamin D could protect placental TC from oxidative stress-induced injury during pregnancy. Since caspase-3 cleavage and ROCK1 activation are key steps of MP formation and release, suppression of caspase-3 cleavage and ROCK1 activation by vitamin D could be a plausible mechanism of vitamin D inhibition of increased MP release induced by oxidative stress.

Submillimeter Diameter Poly(Vinyl Alcohol) Vascular Graft Patency in Rabbit Model.

Microvascular surgery is becoming a prevalent surgical practice. Replantation, hand reconstruction, orthopedic, and free tissue transfer procedures all rely on microvascular surgery for the repair of venous and arterial defects at the millimeter and submillimeter levels. Often, a vascular graft is required for the procedure as a means to bridge the gap between native arteries. While autologous vessels are desired for their bioactivity and non-thrombogenicity, the tedious harvest process, lack of availability, and caliber or mechanical mismatch contribute to graft failure. Thus, there is a need for an off-the-shelf artificial vascular graft that has low thrombogenic properties and mechanical properties matching those of submillimeter vessels. Poly(vinyl alcohol) hydrogel (PVA) has excellent prospects as a vascular graft due to its bioinertness, low thrombogenicity, high water content, and tunable mechanical properties. Here, we fabricated PVA grafts with submillimeter diameter and mechanical properties that closely approximated those of the rabbit femoral artery. In vitro platelet adhesion and microparticle release assay verified the low thrombogenicity of PVA. A stringent proof-of-concept in vivo test was performed by implanting PVA grafts in rabbit femoral artery with multilevel arterial occlusion. Laser Doppler measurements indicated the improved perfusion of the distal limb after implantation with PVA grafts. Moreover, ultrasound Doppler and angiography verified that the submillimeter diameter PVA vascular grafts remained patent for 2 weeks without the aid of anticoagulant or antithrombotics. Endothelial cells were observed in the luminal surface of one patent PVA graft. The advantageous non-thrombogenic and tunable mechanical properties of PVA that are retained even in the submillimeter diameter dimensions support the application of this biomaterial for vascular replacement in microvascular surgery.

Bioequivalence evaluation of new microparticulate capsule and marketed tablet dosage forms of lornoxicam in healthy volunteers

Purpose : To compare oral bioavailability and pharmacokinetic parameters of different lornoxicam formulations and to assess similarity in plasma level profiles by statistical techniques. Methods : An open-label, two-period crossover trial was followed in 24 healthy Pakistani volunteers (22 males, 2 females). Each participant received a single dose of lornoxicam controlled release (CR) microparticles and two doses (morning and evening) of conventional lornoxicam immediate release (IR) tablet formulation. The microparticles were prepared by spray drying method. The formulations were administered again in an alternate manner after a washout period of one week. Pharmacokinetic parameters were determined by Kinetica 4.0 software using plasma concentration-time data. Moreover, data were statistically analyzed at 90 % confidence interval (CI) and Schuirmann’s two one-sided t-test procedure. Results : Peak plasma concentration (C max ) was 20.2 % lower for CR formulation compared to IR formulation (270.90 ng/ml vs 339.44 ng/ml, respectively) while time taken to attain C max (t max ) was 5.25 and 2.08 h, respectively. Area under the plasma drug level versus time (AUC) curve was comparable for both CR and IR formulations. The 90 % confidence interval (CI) values computed for C max , AUC 0-24 , and AUC 0- , after log transformation, were 87.21, 108.51 and 102.74 %, respectively, and were within predefined bioequivalence range (80 - 125 %). Conclusion : The findings suggest that CR formulation of lornoxicam did not change the overall pharmacokinetic properties of lornoxicam in terms of extent and rate of lornoxicam absorption. Keywords : Analgesic, Microparticles, Controlled release, Lornoxicam, NSAIDs, Pharmacokinetics

Abstract 191: Microparticles Released From Various Cell lines are ABCA1 Dependent

The physiological functions of microparticles (MPs) released from cells remain poorly defined. Here, we study MPs released from various cell lines; baby hamster kidney (BHK) cells, human THP-1 and hepG2 cells also expressing ABCA1. To assess the role of MPs in cholesterol homeostasis we incubated cells with apoA-I or CS-6253 (CS) a novel apoE derived mimetic peptide and potential cholesterol efflux and HDL modulator. When incubated with BHK cells expressing ABCA1 under mifepristone and Mock cells in time dependent manner (0,5 to 24h) we found that MPs are ABCA1 dependent. Accordingly the dependence of MPs release on ABCA1 activity is confirmed in human THP-1 cells and hepG2 cells after blocking ABCA1 with probucol (10 μM, 24h). Given the rate-limiting step of ABCA1 in cholesterol homeostasis, we hypothesized that MPs may have a role in cholesterol transport from cells. To address this point we isolated MPs by FPLC from media collected from 3 H-cholesterol labelled cells that were exposed or not to (1 μM) apoA-I, or CS in time dependent manner. We demonstrate that MPs accounted for ~ 1/3 of the total cholesterol released to the medium. In addition, we found that cholesterol carried by MPs is redistributed among resident lipoproteins under conditions when 3 H-MPs were incubated in plasma (1h, 37°C), as shown by FPLC profiles. In addition, the cholesterol regulatory role in cellular release of MPs was tested after depletion of plasma membrane cholesterol by methyl-β-cyclodextrin (CDX, 10 mM, for 30 min). There was a significant decrease in cholesterol counts from MPs released in CDX treated macrophages THP-1 cells when compared to cells not treated relative to each condition: cells alone (16%, p&lt;0.05), cells incubated with; apoA-I (48%, p&lt;0.01), or CS (66%, p&lt;0.0001), indicating that MPs require membrane cholesterol. MPs isolated from cells do not contain apoA-I or CS in contrast to HDL particles as confirmed by Western blotting analysis. MPs characterization by nanoparticle tracking analysis demonstrated vesicles that display (67.76±4 to 920±26nm) average size. We conclude that MPs generation was markedly decreased by blocking ABCA1 suggesting that ABCA1 is critical determinant for MPs formation, and potentially cell communication.

Abstract 564: Inflammasome-induced Endothelial Microparticles Exert Detrimental Effects on Recipient Vascular Cells

Background: The inflammasome, a multi-protein signaling platform, is an important mediator of vascular inflammation resulting in atherosclerosis. Microparticles (MPs) are small membrane vesicles, specifically packaged and released from apoptotic cells for intercellular communication. Endothelial cell derived MPs have recently been linked to atherogenesis. Whether inflammasome activation in endothelial cells mediates MP release and thereby exert biological effects on recipient vascular cells is unknown. Methods and results: Human coronary artery endothelial cells (HCAEC) were primed with 1μg/ml LPS and subsequently stimulated with 20μM Nigericin leading to a specific inflammasome activation as detected by significant upregulation of Caspase-1 mRNA in RT-PCR and protein in Westernblots (Figure1). Inflammasome activation in vascular cells lead to formation of endothelial microparticles (EMP) in a time- and dosis-dependend manner (Figure 2). Comparison of EMP with beads of known size using flow cytometric and electron microscopic imaging could point out an EMP-size between 0,1 – 1 μm (Figure 3). EMP-uptake by recipient vascular cells could be illustrated by fluorescence-microscopic imaging of PKH26 labeled EMP (Figure 4). Viability assay and scratch assay showed detrimental effects of EMPs from inflammasome activated cells on recipient vascular cells. Viability, proliferation and migration were reduced significantly 4h after treatment (Figure 5). Conclusions: We show for the first time that Nigericin, an established inflammasome activator, leads to inflammasome activation and release of microparticles by endothelial cells. Furthermore, that these microparticles are taken up by recipient vascular cells and thereby cause cell death accompanied with reduced cell migration and proliferation.

High-frequency (13.56-MHz) and ultrahigh-frequency (915-MHz) radio identification systems do not affect platelet activation and functions.

In radiofrequency identification (RFID) systems used in labeling of blood components, blood cells are subjected to the direct influence of electromagnetic waves throughout the storage period. The aim of this study was to prove the safety of storage of platelet concentrates (PCs) in containers labeled with RFID tags. Ten pooled PCs obtained from 12 buffy coats each suspended in additive solution were divided into three separate containers that were assigned to three groups: control, PCs labeled with ultrahigh frequency (UHF) range tags and exposed to 915-MHz radio waves, and PCs labeled with high-frequency (HF) range tags and exposed to 13.56-MHz radio waves. PCs were stored at 20 to 24°C for 7 days. In vitro tests of platelet (PLT) function were performed on the first, fifth, and seventh days of storage. There were no significant differences in pH; hypotonic shock resistance; surface expression of CD62P, CD42a, or CD63; release of PLT-derived microparticles; PLT aggregation; and number of PLTs between PCs stored at a constant exposure to radio waves of two different frequencies and the control group on the first, fifth, and seventh days of storage. The results of the study indicate no impact of electromagnetic radiation generated in HF and UHF RFID systems and constant contact with the tags on the quality of stored PCs.

Endothelial, platelet, and macrophage microparticle levels do not change acutely following transcatheter aortic valve replacement.

BackgroundPatients with severe aortic stenosis have increased levels of prothrombotic and proinflammatory microparticles (MP), and MPs actively regulate pathological processes that lead to atherothrombotic cardiovascular events. Shear stress is a validated stimulus of MP production, and abnormal shear stress in aortic stenosis increases MP release in ex-vivo studies. We hypothesized that in patients with severe aortic stenosis, percutaneous replacement of the aortic valve (TAVR) would reduce abnormal shear stress and would decrease levels of circulating MPs.FindingsThe experimental protocol utilized flow cytometry (FC) and nanoparticle tracking analysis (NTA) to quantify circulating plasma MP levels in aortic stenosis patients at baseline and 5 days after TAVR. The baseline and 5 day MP counts measured by FC were 6.10⋅105 ± 1.21⋅105 MP/μL and 5.74⋅105 ± 9.54⋅104 MP/μL, respectively (p = 0.91). The baseline and 5 day MP counts measured by NTA were 9.29⋅1013 ± 1.66⋅1013 MP/μL and 3.95⋅1014 ± 3.11⋅1014 MP/μL, respectively (p = 0.91). When MPs were stratified by cell source, there was no difference in pre/post TAVR endothelial, platelet, or leukocyte MP levels.ConclusionLevels of circulating MPs do not change acutely following TAVR therapy for aortic stenosis.Trial registered at clinicaltrials.gov NCT02193035 on July 11, 2014.Electronic supplementary materialThe online version of this article (doi:10.1186/s12952-016-0051-2) contains supplementary material, which is available to authorized users.

PO-48 - Assessment of the procoagulant potential state of tumour-MP in cancer patients

The venous thromboembolism is considered one of the highest risk factor in cancer patients for instance ovarian and pancreas. This hypercoagulability state is believed to be caused by tumour cells that can produce a variety of procaogulant factors including tissue factor (TF) bearing microparticles (MP). Chemotherapy is an independent risk factor for venous thromboembolism (VTE) in patients and it can leads to coagulation activation and may increase microparticles that can increase risk of thrombosis. Therefore, our current hypothesis is that this increased risk of VTE is due to release of tumour MP into the blood. To further investigate this mechanism an ex-vivo microfluidic model system was developed wherein tumour spheroids were grown and transferred onto a microfluidic chip and assessed, under flow conditions, for procoagulant activity (PCA). Tumour spheroids from pancreatic cancer cell line AsPC1 and human glioblastoma cell line U87 were generated using the liquid overlay method in a 96-well plate, then transferred to a microfluidic chip, designed with a trap within the device to immobilise the spheroid. The procoagulant potential of cell-free supernatant was measured using a prothrombin time clotting assay. Procoagulant activity was assessed under flow rate of 3.0 μL min-1 for 6 hours. Several tumour cell lines (A2780, SKOV3, MIA Paca2, AsPC1 and U87) were assessed for PCA of media (MP associated) and then subsequently assessed for spheroid formation. Prothrombin time of cell-free media was A2780: 794s, SKOV3: 203.4s, MIA Paca2: 412s, AsPC1: 69s and U87: 50.3s. The pancreatic cell line AsPC-1 and glioblastoma cell line U87 were selected for further study on the basis of relatively high PCA and ability to form stable spheroids. When transferred to a microfluidic chip, AsPC1 tumour spheroids showed a slowing of PCA of media over a 6-hour period from 36.6 to 309s. U87 tumour spheroids showed a reduction in PCA of media over a 6-hour period from 51.3 to 108.9s. This is the first report of tumour spheroids maintained in a microfluidic device and then subsequently assessed for PCA. Tumour spheroids of AsPC1 were shown to produce continuous procaogulant activity and this is presumably due to tumour microparticle release. This new model system offers a way to assess tumour associated PCA under flow conditions.

Leptin induces the generation of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells

BackgroundObesity is linked to increased thrombotic risk. Circulating leptin concentration correlates with body mass index. Microparticles are small (.05–1μm) vesicles shed by activated and apoptotic cells, involved in numerous pathophysiologically relevant phenomena including blood coagulation and thrombosis. We tested the hypothesis that leptin induces the shedding of procoagulant, tissue factor bearing microparticles by human peripheral blood mononuclear cells, and investigated the intracellular mechanisms leading to microparticle release upon incubation with leptin. MethodsPeripheral blood mononuclear cells were isolated from healthy donors. Cells were incubated with leptin in the presence or in the absence of a phospholipase C inhibitor, U73122, a calmodulin inhibitor, W-7, and three inhibitors of mitogen activated protein kinases. Microparticle generation was assessed as phosphatidylserine concentration with a prothrombinase assay and by cytofluorimetric analysis. Tissue factor expression on microparticles was measured with a one-stage clotting assay. Intracellular calcium concentration was assessed by a fluorescent probe. ResultsLeptin increased intracellular calcium mobilization and stimulated the generation of tissue factor-bearing MP by peripheral blood mononuclear cells, as assessed by phosphatidylserine quantification, clotting tests and flow-cytometry. U73122, PD98059 (an extracellular signal-regulated kinase1/2 inhibitor), and W-7, significantly inhibited leptin-induced MP release. SB203580 (a p38 inhibitor), and SP600125 (a c-Jun N-terminal kinase inhibitor) had no effect. ConclusionLeptin-induced generation of procoagulant microparticles might represent a link between obesity and atherothrombotic risk. Inhibition of leptin-induced microparticle generation might prove a viable strategy for the reduction of such risk in obese individuals.

N-Acetylcysteine Amide Protects Against Oxidative Stress-Induced Microparticle Release From Human Retinal Pigment Epithelial Cells.

PurposeOxidative stress is a major factor involved in retinal pigment epithelium (RPE) apoptosis that underlies AMD. Drusen, extracellular lipid- and protein-containing deposits, are strongly associated with the development of AMD. Cell-derived microparticles (MPs) are small membrane-bound vesicles shed from cells. The purpose of this study was to determine if oxidative stress drives MP release from RPE cells, to assess whether these MPs carry membrane complement regulatory proteins (mCRPs: CD46, CD55, and CD59), and to evaluate the effects of a thiol antioxidant on oxidative stress–induced MP release.MethodsRetinal pigment epithelium cells isolated from human donor eyes were cultured and treated with hydrogen peroxide (H2O2) to induce oxidative stress. Isolated MPs were fixed for transmission electron microscopy or processed for component analysis by flow cytometry, Western blot analysis, and confocal microscopy.ResultsTransmission electron microscopy showed that MPs ranged in diameter from 100 to 1000 nm. H2O2 treatment led to time- and dose-dependent elevations in MPs with externalized phosphatidylserine and phosphatidylethanolamine, known markers of MPs. These increases were strongly correlated to RPE apoptosis. Oxidative stress significantly increased the release of mCRP-positive MPs, which were prevented by a thiol antioxidant, N-acetylcysteine amide (NACA).ConclusionsThis is the first evidence that oxidative stress induces cultured human RPE cells to release MPs that carry mCRPs on their surface. The levels of released MPs are strongly correlated with RPE apoptosis. N-acetylcysteine amide prevents oxidative stress–induced effects. Our findings indicate that oxidative stress reduces mCRPs on the RPE surface through releasing MPs.

Erythrocyte and platelet proteomics in hematological disorders.

Erythrocytes undergo ineffective erythropoesis, hemolysis, and premature eryptosis in sickle cell disease and thalassemia. Abnormal hemoglobin variants associated with hemoglobinopathy lead to vesiculation, membrane instability, and loss of membrane asymmetry with exposal of phosphatidylserine. This potentiates thrombin generation resulting in activation of the coagulation cascade responsible for subclinical phenotypes. Platelet activation also results in the release of microparticles, which express and transfer functional receptors from platelet membrane, playing key roles in vascular reactivity and activation of intracellular signaling pathways. Over the last decade, proteomics had proven to be an important field of research in studies of blood and blood diseases. Blood cells and its fluidic components have been proven to be easy systems for studying differential expressions of proteins in hematological diseases encompassing hemoglobinopathies, different types of anemias, myeloproliferative disorders, and coagulopathies. Proteomic studies of erythrocytes and platelets reported from several groups have highlighted various factors that intersect the signaling networks in these anucleate systems. In this review, we have elaborated on the current scenario of anucleate blood cell proteomes in normal and diseased individuals and the cross-talk between the two major constituent cell types of circulating blood.

Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.

Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance: This study demonstrates the regulation of chondrogenesis and osteogenesis with regard to endochondral bone formation in high-density stem cell systems through the controlled presentation of inductive factors from incorporated microparticles. This work lays the foundation for a rapidly implantable tissue engineering system that promotes bone repair via endochondral ossification, a pathway that can delay the need for a functional vascular network and has an intrinsic ability to promote angiogenesis. The modular nature of this system lends well to using different cell types and/or growth factors to induce endochondral bone formation, as well as the production of other tissue types.

Thrombotic Role of Blood and Endothelial Cells in Uremia through Phosphatidylserine Exposure and Microparticle Release.

The mechanisms contributing to an increased risk of thrombosis in uremia are complex and require clarification. There is scant morphological evidence of membrane-dependent binding of factor Xa (FXa) and factor Va (FVa) on endothelial cells (EC) in vitro. Our objectives were to confirm that exposed phosphatidylserine (PS) on microparticle (MP), EC, and peripheral blood cell (PBC) has a prothrombotic role in uremic patients and to provide visible and morphological evidence of PS-dependent prothrombinase assembly in vitro. We found that uremic patients had more circulating MP (derived from PBC and EC) than controls. Additionally, patients had more exposed PS on their MPs and PBCs, especially in the hemodialysis group. In vitro, EC exposed more PS in uremic toxins or serum. Moreover, reconstitution experiments showed that at the early stages, PS exposure was partially reversible. Using confocal microscopy, we observed that PS-rich membranes of EC and MP provided binding sites for FVa and FXa. Further, exposure of PS in uremia resulted in increased generation of FXa, thrombin, and fibrin and significantly shortened coagulation time. Lactadherin, a protein that blocks PS, reduced 80% of procoagulant activity on PBC, EC, and MP. Our results suggest that PBC and EC in uremic milieu are easily injured or activated, which exposes PS and causes a release of MP, providing abundant procoagulant membrane surfaces and thus facilitating thrombus formation. Blocking PS binding sites could become a new therapeutic target for preventing thrombosis.

Abstract 15426: Inflammasome-induced Signalling Mechanisms via Microparticles

Introduction: The inflammasome, a multi-protein signaling platform, has been shown to be an important mediator in the development of atherogenesis. Microparticles (MPs) are small membrane vesicles, which are released from apoptotic cells. Whether inflammasome activation leads to the release of MPs by vascular cells is unknown. Hypothesis: Inflammasome-activation in vascular cells leads to microparticle release. These microparticles contain components of the inflammasome and exert functional effects on other vascular cells. Methods: EMP (endothelial microparticles) and MMP (monocytic microparticles) were quantified by flow cytometry using TruCount tubes. Inflammasome-activation was investigated by IL-1β ELISA and immunoblotting. The latter was also used to determine EMP content. Viability Assay was performed to test cell-viability and Scratch assay to examine effects of EMP on migration and proliferation of vascular cells. Results: Human coronary artery endothelial cells (HCAEC) and monocytes were primed with LPS and subsequently stimulated with either ATP or Nigericin, two established inflammasome activators. MP release was quantified by flow cytometry using TruCount tubes. Stimulation of HCAEC and monocytes with both Nigericin and ATP resulted in a time-dependent release of MPs. Inflammasome-activation in HCAEC was confirmed by the release of IL-1β into the cell supernatant and immunoblotting against Caspase-1. Immunoblotting also showed that inflammasom-activated HCAEC and the associated EMP contain pro-Caspase-1, active Caspase-1 and NALP-3. Inoculation of HCAEC with EMP, derived from HCAEC stimulated with Nigericin, lead to a rapid and significant cell death and reduced cell migration/ proliferation shown by Viability Assay and Scratch Assay, respectively. Conclusions: We show for the first time that Nigericin and ATP, two established inflammasome activators, lead to inflammasome activation and release of microparticles by vascular cells. Furthermore, we could demonstrate that these microparticles contain pro-Caspase-1, mature-Caspase-1 and NALP-3 and that they cause cell death accompanied with reduced cell migration and proliferation.

Indolic uremic solutes enhance procoagulant activity of red blood cells through phosphatidylserine exposure and microparticle release.

Increased accumulation of indolic uremic solutes in the blood of uremic patients contributes to the risk of thrombotic events. Red blood cells (RBCs), the most abundant blood cells in circulation, may be a privileged target of these solutes. However, the effect of uremic solutes indoxyl sulfate (IS) and indole-3-acetic acid (IAA) on procoagulant activity (PCA) of erythrocyte is unclear. Here, RBCs from healthy adults were treated with IS and IAA (mean and maximal concentrations reported in uremic patients). Phosphatidylserine (PS) exposure of RBCs and their microparticles (MPs) release were labeled with Alexa Fluor 488-lactadherin and detected by flow cytometer. Cytosolic Ca2+ ([Ca2+]) with Fluo 3/AM was analyzed by flow cytometer. PCA was assessed by clotting time and purified coagulation complex assays. We found that PS exposure, MPs generation, and consequent PCA of RBCs at mean concentrations of IS and IAA enhanced and peaked in maximal uremic concentrations. Moreover, 128 nM lactadherin, a PS inhibitor, inhibited over 90% PCA of RBCs and RMPs. Eryptosis or damage, by indolic uremic solutes was due to, at least partially, the increase of cytosolic [Ca2+]. Our results suggest that RBC eryptosis in uremic solutes IS and IAA plays an important role in thrombus formation through releasing RMPs and exposing PS. Lactadherin acts as an efficient anticoagulant in this process.