Plasma membrane microparticles in angiogenesis: role in ischemic diseases and in cancer

Microparticles are small fragments of the plasma membrane released by activated and/or apoptotic cells. In theory, all type of cells can shed microparticles representing a physiological process in the cell life. Mainly, microparticles generation has been studied in different cardiovascular pathologies due to the facility to obtain blood samples from individuals. Although microparticles have been considered as simply markers of several diseases, in the last decade, several studies support the hypothesis that they participate in the regulation of the cardiovascular system function by carrying biological messages between cells. Among the effects of microparticles, recent data show that they can be implicated in the modulation of neovascularization, an essential function of cells from cardiovascular system during either ischemic diseases or cancer development. Whereas during pathologies associated with ischemia an increase of neovascularization may have beneficial effects, anti-angiogenic strategies represent new approaches for manipulation of tumor development. Here, we give an overview of the mechanisms and factors involved in neovascularization, and finally, we look at the role and the consequences of the modulation of this process by microparticles in pathological situations.     

Induction of type I IFN is a physiological immune reaction to apoptotic cell-derived membrane microparticles

Membrane microparticles (MMP) released from apoptotic cells deliver signals that secure the anti-inflammatory response beyond the nearest proximity of the apoptotic cell. Plasmacytoid dendritic cells (pDC) are sentinels prepared to detect cellular processes that endanger the organism. They play a key role in the regulation of both pro- and anti-inflammatory immune responses. Based on the assumption that pDC could participate in the initiation of the anti-inflammatory response to apoptotic cells, we investigated the effects of apoptotic cell-derived MMP on human pDC. The results obtained in our experiments confirmed that MMP released from apoptotic cells trigger IFN-α secretion from human pDC. They further suggest that pDC activation results from sensing of DNA contained in MMP. MMP-DNA displays a particularly strong stimulatory activity compared with MMP-RNA and other sources of DNA. Inhibition of MMP-induced IFN-α secretion by cytochalasin D, chloroquine, and an inhibitory G-rich oligodeoxynucleotide identify TLR9 as the receptor for MMP-DNA. In marked contrast to the pDC response in autoimmune patients, in healthy subjects MMP-mediated stimulation of pDC-derived IFN-α was found to be independent of FcγRIIA (CD32A). Based on our findings, we conclude that induction of pDC-derived IFN-α by MMP is a physiological event; future investigations are necessary to elucidate whether pDC activation promotes inflammation or propagates tolerance in the context of apoptotic cell clearance.     

The release of microparticles by apoptotic cells and their effects on macrophages

Microparticles are small membrane vesicles released from the cell membrane by exogenous budding. To elucidate the interactions of microparticles with macrophages, the effect of microparticles released from Jurkat T cells on RAW 264.7 cells was determined. Microparticles were isolated by differential centrifugation, using FACS analysis with annexin V and cell surface markers for identification. Various inducers of apoptosis increased the release of microparticles from Jurkat cells up to 5-fold. The released microparticles were then cultured with RAW 264.7 cells. As shown by confocal microscopy and FACS analysis, RAW 264.7 macrophages cleared microparticles by phagocytosis. In addition, microparticles induced apoptosis in RAW 264.7 cells in a dose-dependent manner with up to a 5-fold increase of annexin V positive cells and 9-fold increase in caspase 3 activity. Cell proliferation as determined by the MTT test was also reduced. Furthermore, microparticles stimulated the release of microparticles from macrophages. These effects were specific for macrophages, since no apoptosis was observed in NIH 3T3 and L929 cells. These findings indicate that microparticles can induce macrophages to undergo apoptosis, in turn resulting in a further increase of microparticles. The release of microparticles from apoptotic cells may therefore represent a novel amplification loop of cell death.     

Microparticle‐mediated gene delivery for the enhanced expression of a 19‐kDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl‐terminal fragment of merozoite surface protein 1 (MSP1₁₉) is a major component of the invasion‐inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic‐co‐glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP1₁₉ is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h^?1. High levels of gene expression and moderate cytotoxicity in COS‐7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z‐average hydrodynamic diameters of 1.50–2.10 μm and zeta potentials of 17.8–23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA‐mediated microparticles can be employed as potential gene delivery systems to antigen‐presenting cells in the prevention of malaria. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Microparticles as biomarkers in autoimmunity: from dust bin to center stage

Microparticles are small membrane-bound vesicles released from activated and dying cells. As shown in a study of primary Sjogren's syndrome, systemic lupus erythematosus and rheumatoid arthritis, levels of microparticles in the blood, as measured by a solid-phase prothrombinase assay or flow cytometry, are increased with autoimmunity. Among patients with these conditions, however, particle numbers were inversely related to disease activity and levels of the enzyme secretory phospholipase A₂ that can digest membrane lipids and perhaps cause particle loss. These findings suggest microparticles as novel biomarkers for autoimmunity, with levels reflecting events leading to their loss as well as production.     

Human plasma membrane-derived vesicles inhibit the phagocytosis of apoptotic cells - Possible role in SLE

Plasma membrane-derived vesicles (PMVs) also known as microparticles, are small membrane-bound vesicles released from the cell membrane via blebbing and shedding. PMVs have been linked with various physiological functions as well as pathological conditions such as inflammation, autoimmune disease and cardiovascular disease. PMVs are characterised by the expression of phosphatidylserine (PS) on the plasma membrane. PS, also expressed on apoptotic cells (ACs) enables macrophages to phagocytose ACs. As it is widely known that PMV production is increased during apoptosis, we were able to show that PMVs could compete dose dependently with ACs for the PS receptor on macrophages, so reducing phagocytosis of ACs. In a clinical setting this may result in secondary necrosis and further pathological conditions. In SLE in which there are raised PMV levels, there is an anti-phospholipid-mediated increase in PMV release, which can be abrogated by depletion of IgG. Our work provides an insight into how PMVs may play a role in the aetiology of autoimmune disease, in particular SLE.     

Isolation and identification of potential urinary microparticle biomarkers of bladder cancer

Bladder cancer leads to approximately 13,000 deaths annually in the United States. Early disease is often treated with minimal morbidity and has good prognosis, while the opposite is true for advanced disease. Currently, no tools exist for early detection of this cancer. Microparticles are small, subcellular particles released by essentially all cells upon activation and are known to be produced constitutively by cancer cells. Since most bladder cancers originate in the urothelial cells lining the lumen of the organ, we hypothesize that these cells will release microparticles into the urine. The goal of this study was to identify potential biomarkers in the urinary microparticles of individuals with bladder cancer. Urine microparticles from five healthy individuals and four individuals with bladder cancer were isolated. Samples were delipidated by PAGE and trypsin-digested, peptides were extracted, and the proteome was examined by LC-MS/MS using a Thermo Finnigan LTQ and LTQ-FT ion trap mass spectrometer. Protein identification was determined by SEQUEST and relative quantitation was assessed by comparing spectral counts. Eight proteins were elevated in the microparticles from individuals with bladder cancer. They include five proteins associated with the epidermal growth factor receptor pathway, the alpha subunit of GsGTP binding protein, resistin, and retinoic acid-induced protein 3. Further studies will be needed to validate these potential biomarkers.     

Are prostaglandins involved in the regulation of coronary blood flow? A review of the evidence

Prostaglandins (PG's) fulfil most of the criteria required for the metabolic "coupler" linking increases in myocardial activity with increases in coronary blood flow. They are synthesized by the heart and coronary vessels, profoundly modify coronary blood flow in low concentrations and are released under conditions of hypoxia and myocardial ischaemia. Studies with inhibitors of PG-synthetase however provide no firm evidence that PG's are involved in the physiological regulation of myocardial blood flow. These studies are reviewed.     

Monitoring RNA release from human rhinovirus by dynamic force microscopy

Human rhinoviruses were imaged under physiological conditions by dynamic force microscopy. Topographical images revealed various polygonal areas on the surfaces of the 30-nm viral particles. RNA release was initiated by exposure to a low-pH buffer. The lengths of the RNAs that were released but still connected to the virus capsid varied between 40 and 330 nm, whereas RNA molecules that were completely released from the virus were observed with lengths up to 1 micro m. Fork-like structure elements with 30-nm extensions were sometimes resolved at one end of the RNA molecules. They possibly correspond to the characteristic multi-stem-loop conformation, the internal ribosomal entry site, located at the 5' region of the genome. This study demonstrates that dynamic force microscopy can be used to study viral RNA release in situ under physiological conditions.     

Cytometric analysis of high shear-induced platelet microparticles and effect of cytokines on microparticle generation

BACKGROUND: Microparticles released from platelets may play a role in the normal hemostatic response to vascular injury, because they exhibit prothrombinase activity. Microparticles are generated by high shear stress and may be formed in diseased small arteries and arterioles in various clinical settings. However, the surface composition of high shear-induced platelet microparticles is unknown. It was recently shown that some cytokines modulate platelet activation. However, no reports are available concerning the effect of cytokines on high shear-induced platelet aggregation (SIPA) microparticle generation. MATERIALS AND METHODS: Measurement of SIPA was performed with a cone-plate viscometer. The conformational characteristics of high shear (108 dynes/cm(2))-induced platelet microparticles were analyzed by flow cytometry and confocal laser scanning microscopy. Effects of cytokines for high SIPA microparticle generation were also analyzed using flow cytometry. RESULTS: The overall pattern of monoclonal antibody binding in high shear-induced microparticles was almost the same as that in activated platelets under high shear stress. Microparticles exhibited markedly increased Annexin V binding. In fluorescent confocal images, small and fine regions of fluorescence (microparticles) were recognized separate from platelet fluorescence. Thrombopoietin not only induced platelet activation, as demonstrated by CD62P expression, but also increased the number of microparticles. Erythropoietin and interleukin-6 enhanced only microparticle generation. CONCLUSIONS: These results suggest that microparticles possessing procoagulant activity are released by platelet activation when levels of certain cytokines increase under high shear stress in various clinical settings.     

内皮细胞微粒与脓毒症研究进展

内皮细胞微粒是活化或凋亡的内皮细胞表面释放的直径1μm的小囊泡。它是反映内皮功能的标志物。研究表明在脓毒症的发生发展过程中,内皮细胞微粒在炎症反应、凝血反应、血管内皮功能等多方面能发挥有利和有害双方面的作用。脓毒症的研究进展和内皮细胞微粒密切相关。该文将就内皮细胞微粒与脓毒症研究进展做一简要综述。     

T lymphocytes export proteasomes by way of microparticles: a possible mechanism for generation of extracellular proteasomes

The 20S proteasome is almost exclusively localized within cells. High levels of extracellular proteasomes are also found circulating in the blood plasma of patients suffering from a variety of inflammatory, autoimmune and neoplastic diseases. However, the origin of these proteasomes remained enigmatic. Since the proteome of microparticles, small membrane enclosed vesicles released from cells, was shown to contain proteasomal subunits, we studied whether intact proteasomes are actively released into the extracellular space. Using human primary T lymphocytes stimulated with CaCl₂ and the calcium ionophore A23187 to induce membrane blebbing we demonstrate that microparticles contain proteolytically active 20S proteasomes as well as the proteasome activator PA28 and subunits of the 19S proteasome regulator. Furthermore, our experiments reveal that incubation of in vitro generated T lymphocyte‐microparticles with sphingomyelinase results in the hydrolysis of the microparticle membranes and subsequent release of proteasomes from the vesicles. Thus, we here show for the first time that functional proteasomes can be exported from activated immune cells by way of microparticles, the dissolution of which may finally lead to the generation of extracellular proteasomes.     

Mucoadhesive polyelectrolyte microparticles containing recombinant human insulin and its analogs aspart and lispro

Microparticles containing recombinant human insulin and its analogs aspart and lispro were prepared using an alternate adsorption of chitosan and dextran sulfate from solutions onto microaggregates of protein-dextran sulfate insoluble complex. The following properties of polyelectrolyte hormone-containing microparticles were studied: pH stability, surface charge, mucoadhesive properties, Ca²⁺ binding, degradation under the influence of proteases (trypsin, chymotrypsin). The influence of the self-association ability of encapsulated insulins on the form of protein releasing from microparticles was studied. Insulins aspart and lispro released from the microparticles as monomers were more liable to proteolysis than human insulin released as a hexamer. The combined effect of properties of polyelectrolyte microparticles and of encapsulated recombinant proteins on the bioavailability of insulin under peroral administration is discussed.     

Red cell PMVs, plasma membrane-derived vesicles calling out for standards

Plasma membrane-derived vesicles (PMVs) or microparticles are vesicles (0.1-1 μm in diameter) released from the plasma membrane of all blood cell types under a variety of biochemical and pathological conditions. PMVs contain cytoskeletal elements and some surface markers from the parent cell but lack a nucleus and are unable to synthesise macromolecules. They are also defined on the basis that in most cases PMVs express varying amounts of the cytosolic leaflet lipid phosphatidylserine, which is externalised during activation on their surface. This marks the PMV as a biologically distinct entity from that of its parent cell, despite containing surface markers from the original cell, and also explains its role in events such as phagocytosis and thrombosis. There is currently a large amount of variation between investigators with regard to the pre-analytical steps employed in isolating red cell PMVs or RPMVs (which are slightly smaller than most PMVs), with key differences being centrifugation and sample storage conditions, which often leads to result variability. Unfortunately, standardization of preparation and detection methods has not yet been achieved. This review highlights and critically discusses the variables contributing to differences in results obtained by investigators, bringing to light numerous studies of which RPMVs have been analysed but have not yet been the subject of a review.     

Characterisation and properties of ectosomes released by human polymorphonuclear neutrophils

Human neutrophils release vesicles when activated in vitro and in vivo, in local and systemic inflammation. We have suggested that the presence of these vesicles is due to ectocytosis, defined as the release of rightside-out oriented vesicles expressing a select set of membrane proteins. Herein we have characterised the vesicles released by neutrophils to be ectosomes with specific properties. They contained cytosolic F-actin indicating their outside-out orientation. They bound Annexin V, suggesting that they expose phosphatidylserine, similarly to platelet microparticles. They expressed a subset of cell surface proteins (selectins and integrins, complement regulators, HLA-1, FcgammaRIII, and CD66b, but not CD14, FcgammaRII, and CD87). There was no specificity for transmembrane or glycosyl-phosphatidylinositol-linked proteins and, unexpectedly, L-selectin, known to be cleaved from the surface of activated neutrophils, was present. Ectosomes exposed active enzymes released by neutrophils upon degranulation (matrix metalloproteinase-9, myeloperoxidase, proteinase 3, and elastase). In particular, released myeloperoxidase was able to bind back to ectosomes. The purified complement protein C1q and C1q from serum bound to ectosomes as well. Another aspect of ectosomes was that they became specifically adherent to monocytic and endothelial cells. These observations suggest that neutrophil-derived ectosomes have unique characteristics that make them candidates for playing roles in inflammation and cell signaling.     

三氧化二砷对急性早幼粒细胞白血病细胞微粒数量及促凝活性的影响

目的:观察急性早幼粒细胞白血病(APL)细胞来源微粒(APL-MP)的促凝活性、表面组织因子(TF)表达情况、TF在其促凝活性中发挥的作用及分化治疗药物三氧化二砷(ATO)对上述指标有何影响。方法:选取3例初发APL患者,提取骨髓APL细胞,3名缺铁性贫血患者提取骨髓单个核细胞作为对照。分别用不同浓度ATO处理APL细胞24 h、48 h、72 h,收集细胞培养液提取微粒。采用流式细胞术对微粒进行定量分析并进行微粒表面TF表达情况检测;利用凝血实验比较不同组细胞释放微粒的促凝血活性;应用抗TF抗体抑制微粒促凝血活性实验检测TF在APL-MP的促凝血活性中发挥多大作用。结果:1.0μM及2.0μM ATO能显著促进APL细胞释放微粒。与正常骨髓来源单个核细胞释放的微粒相比,骨髓APL-MP的TF表达及促凝活性均显著增高,0.5μM及1.0μM ATO处理可以有效降低APL-MP的TF表达及促凝活性,且这一作用呈时间依赖性。各组APL-MP经抗TF抗体孵育后凝血时间显著延长。结论:APL-MP的TF表达和促凝学活性均显著增高,并且TF在APL-MP的促凝血活性中发挥着重要作用。ATO能显著促进APL细胞释放微粒,低浓度ATO可以有效降低APL-MP的TF表达及促凝血活性。     

Thrombin-induced platelet microparticles improved the aggregability of cryopreserved platelets

Platelets were activated with freezing/thawing and thrombin stimulation, and platelet microparticles generated following platelet activation were isolated with ultracentrifugation. The effects of platelet microparticles on platelet activation were studied with annexin V assay, protein tyrosine phosphorylation, and platelet aggregation. Freezing-induced platelet microparticles decreased but thrombin-induced platelet microparticles increased platelet annexin V binding and aggregation. Freshly washed platelets were cryopreserved using epinephrine and dimethyl sulfoxide (Me(2)SO) as combined cryoprotectants, and stimulated with thrombin-induced platelet microparticles. Following incubation of thrombin-induced platelet microparticles, the reaction time of platelets to agonists decreased but the percentages of aggregation increased, such as washed platelets from 44% +/- 30 to 92% +/- 7, p < 0.001, and cryopreserved platelets from 66% +/- 10 to 77% +/- 7, p < 0.02. By increasing platelet aggregability, platelet microparticles recovered after thrombin stimulation improved platelet function for transfusion. A 53-kDa platelet microparticle protein showed little phosphorylation if it was released from resting platelets or platelets stimulated with ADP, epinephrine, propyl gallate or dephosphorylation if it was derived from ionophore A 23187-stimulated platelets. However, the same protein released from frozen platelets showed significant tyrosine phosphorylation. Since a microparticle protein with 53 kDa was compatible with protein tyrosine phosphatase-1B (PTP-1B), its phosphorylation suggests the inhibition of enzyme activity. The microparticle proteins derived from thrombin-stimulated platelets were significantly phosphorylated at 64 kDa and pp60c-src, suggesting that the activation of tyrosine kinases represents a possible mechanism of thrombin-induced platelet microparticles to improve platelet aggregation.     

Fully acid-degradable biocompatible polyacetal microparticles for drug delivery

A library of polyurethanes and polyureas with different hydrophobicities containing the same acid-degradable dimethyl ketal moiety embedded in the polymer main chain have been prepared. All polymers were synthesized using an AA-BB type step-growth polymerization by reaction of bis(p-nitrophenyl carbamate/carbonate) or diisocyanate monomers with an acid-degradable, ketal-containing diamine. These polymers were designed to hydrolyze at different rates in mildly acidic conditions as a function of their hydrophobicity to afford small molecules only with no polymeric byproduct. The library of polymers was screened for the formation of microparticles using a double emulsion technique. The microparticles that were obtained degraded significantly faster at acidic pH (5.0) than at physiological pH (7.4) with degradation kinetics related to the hydrophobicity of the starting polymer. In vitro studies demonstrated the ability of the FITC-BSA loaded microparticles to be phagocytosed by macrophages resulting in a 10-fold increase in the protein uptake compared to a free protein control; in addition, the microparticles were found to be nontoxic at the concentrations tested of up to 1 mg/mL. The ease of preparation of the polymers coupled with the ability to tune their hydrophobicity and the high acid sensitivity of the microparticles identify this new class of materials as promising candidates for the delivery of bioactive materials.     

Effects of glycerol on human adipose tissue triglyceride lipase activity

Glycerol fully protects the human adipose tissue triglyceride lipase against the denaturing effects of high and low temperatures. Under such protection, storage of crude preparations at -10 degrees C or incubation at 50 degrees C resulted in a 1.5-3-fold increase of the measured lipase activity. This increase was shown to be related to enzyme newly released from tissular microparticles present in the samples. Advantage may be taken of these observations to improve greatly the conditions of extraction and storage of this lipase activity.