Microparticles engineered to highly express peroxisome proliferator-activated receptor-? decreased inflammatory mediator production and increased adhesion of recipient monocytes.
ABSTRACT: Circulating blood microparticles are submicron vesicles released primarily by megakaryocytes and platelets that act as transcellular communicators. Inflammatory conditions exhibit elevated blood microparticle numbers compared to healthy conditions. Direct functional consequences of microparticle composition, especially internal composition, on recipient cells are poorly understood. Our objective was to evaluate if microparticle composition could impact the function of recipient cells, particularly during inflammatory provocation. We therefore engineered the composition of megakaryocyte culture-derived microparticles to generate distinct microparticle populations that were given to human monocytes to assay for influences recipient cell function. Herein, we tested the responses of monocytes exposed to either control microparticles or microparticles that contain the anti-inflammatory transcription factor, peroxisome proliferator-activated receptor-? (PPAR?). In order to normalize relative microparticle abundance from two microparticle populations, we implemented a novel approach that utilizes a Nanodrop Spectrophotometer to assay for microparticle density rather than concentration. We found that when given to peripheral blood mononuclear cells, microparticles were preferentially internalized by CD11b+ cells, and furthermore, microparticle composition had a profound functional impact on recipient monocytes. Specifically, microparticles containing PPAR? reduced activated monocyte production of the proinflammatory cytokines interleukin-8 and monocyte chemotactic protein-1 compared to activated monocytes exposed to control microparticles. Additionally, treatment with PPAR? microparticles greatly increased monocyte cell adherence. This change in morphology occurred simultaneously with increased production of the key extracellular matrix protein, fibronectin and increased expression of the fibronectin-binding integrin, ITGA5. PPAR? microparticles also changed monocyte mRNA levels of several genes including those under PPAR? control. Overall, the delivery of PPAR? from microparticles to human monocytes influenced gene expression, decreased inflammatory mediator production and increased monocyte adherence. These results support the concept that the composition of blood microparticles has a profound impact on the function of cells with which they interact, and likely plays a role in vascular inflammation.
Project description:The objective of this work was to study the ability of blood cells and their microparticles to transport monomeric and pentameric forms of C-reactive protein (mCRP and pCRP) in the blood of patients with coronary artery disease (CAD). Blood was obtained from 14 patients with CAD 46 ± 13 years old and 8 healthy volunteers 49 ± 13.6 years old. Blood cells and microparticles with mCRP and pCRP on their surface were detected by flow cytometry. Messenger RNA (mRNA) of CRP was extracted from peripheral blood monocytes stimulated with lipopolysaccharide (LPS) and granulocyte-macrophage colony-stimulating factor (GM-CSF). mRNA of CRP in monocytes was detected with PCR. Monocytes were predominantly pCRP-positive (92.9 ± 6.8%). mCRP was present on 22.0 ± 9.6% of monocyte-derived exosomes. mCRP-positive leukocyte-derived microparticle counts were significantly higher (8764 ± 2876/µL) in the blood of patients with CAD than in healthy volunteers (1472 ± 307/µL). LPS and GM-CSF stimulated monocytes expressed CRP mRNA transcripts levels (0.79 ± 0.73-fold), slightly lower relative to unstimulated hepatocytes of the HepG2 cell line (1.0 ± 0.6-fold), but still detectable. The ability of monocytes to transport pCRP in blood flow, and monocyte-derived exosomes to transmit mCRP, may contribute to the maintenance of chronic inflammation in CAD.
Project description:BACKGROUND:Microparticles are submicrometer vesicles that contain RNA and protein derived from their parent cells. Platelet and megakaryocyte microparticles represent 80% of circulating microparticles, and their numbers are elevated in diseases such as cancer and type 2 diabetes. The ability of microparticles to transport protein, lipid and RNA to target cells, as a means of transcellular communication, remains poorly understood. Determining the influence that microparticles have on circulating cells is essential for understanding their role in health and in disease. OBJECTIVES:To develop a novel approach to modify the composition of platelet microparticles, and understand how such changes impact their transcellular communication. METHODS:This novel model utilizes a lentiviral technology to alter the transcription factor peroxisome proliferator-activated receptor-? (PPAR?) content of megakaryoblastic cell lines and primary megakaryocytes, and also the protein composition of generated platelets and microparticles. The subsequent microparticles were isolated and added to target cells for assessment of uptake and resultant signaling events. RESULTS:We successfully engineered microparticles to contain green fluorescent protein and elevated levels of PPAR?. We found that these altered microparticles could be internalized by the monocytic cell line THP-1 and primary human microvascular endothelial cells. Importantly, microparticle-delivered PPAR? was shown to increase the expression of fatty acid-binding protein 4 (FABP4), which is a known PPAR? target gene in THP-1 cells. CONCLUSION:This proof-of-concept modification of megakaryocyte, platelet and microparticle composition and subsequent change in target cell physiology is an important new tool to address transcellular communication of microparticles.
Project description:Platelets shed microparticles not only upon activation, but also upon ageing by an apoptosis-like process (apoptosis-induced platelet microparticles, PM(ap)). While the activation-induced microparticles have widely been studied, not much is known about the (patho)physiological consequences of PM(ap) formation. Flow cytometry and scanning electron microscopy demonstrated that PM(ap) display activated integrins and interact to form microparticle aggregates. PM(ap) were chemotactic for monocytic cells, bound to these cells, an furthermore stimulated cell adhesion and spreading on a fibronectin surface. After prolonged incubation, PM(ap) promoted cell differentiation, but inhibited proliferation. Monocyte membrane receptor analysis revealed increased expression levels of CD11b (integrin ?(M)?(2)), CD14 and CD31 (platelet endothelial cell adhesion molecule-1), and the chemokine receptors CCR5 and CXCR4, but not of CCR2. This indicated that PM(ap) polarized the cells into resident M2 monocytes. Cells treated with PM(ap) actively consumed oxidized low-density lipoprotein (oxLDL), and released matrix metalloproteinases and hydrogen peroxide. Further confirmation for the differentiation towards resident professional phagocytes came from the finding that PM(ap) stimulated the expression of the (ox)LDL receptors, CD36 and CD68, and the production of proinflammatory and immunomodulating cytokines by monocytes. In conclusion, interaction of PM(ap) with monocytic cells has an immunomodulating potential. The apoptotic microparticles polarize the cells into a resident M2 subset, and induce differentiation to resident professional phagocytes.
Project description:Ischemic stroke has shown to induce platelet and endothelial microparticle shedding, but whether stroke induces microparticle shedding from additional blood and vascular compartment cells is unclear. Neural precursor cells have been shown to replace dying neurons at sites of brain injury; however, if neural precursor cell activation is associated to microparticle shedding, and whether this activation is maintained at long term and associates to stroke type and severity remains unknown. We analyzed neural precursor cells and blood and vascular compartment cells microparticle shedding after an acute ischemic stroke.Forty-four patients were included in the study within the first 48h after the onset of stroke. The cerebral lesion size was evaluated at 3-7 days of the stroke. Circulating microparticles from neural precursor cells and blood and vascular compartment cells (platelets, endothelial cells, erythrocytes, leukocytes, lymphocytes, monocytes and smooth muscle cells) were analyzed by flow cytometry at the onset of stroke and at 7 and 90 days. Forty-four age-matched high cardiovascular risk subjects without documented vascular disease were used as controls.Compared to high cardiovascular risk controls, patients showed higher number of neural precursor cell- and all blood and vascular compartment cell-derived microparticles at the onset of stroke, and after 7 and 90 days. At 90 days, neural precursor cell-derived microparticles decreased and smooth muscle cell-derived microparticles increased compared to levels at the onset of stroke, but only in those patients with the highest stroke-induced cerebral lesions.Stroke increases blood and vascular compartment cell and neural precursor cell microparticle shedding, an effect that is chronically maintained up to 90 days after the ischemic event. These results show that stroke induces a generalized blood and vascular cell activation and the initiation of neuronal cell repair process after stroke. Larger cerebral lesions associate with deeper vessel injury affecting vascular smooth muscle cells.
Project description:<h4>Background & aims</h4>Ischemia-reperfusion injury (IRI) can cause hepatic failure after liver surgery or transplantation. IRI causes oxidative stress, which injures sinusoidal endothelial cells (SECs), leading to recruitment and activation of Kupffer cells, platelets and microcirculatory impairment. We investigated whether injured SECs and other cell types release microparticles during post-ischemic reperfusion, and whether such microparticles have pro-inflammatory, platelet-activating and pro-injurious effects that could contribute to IRI pathogenesis.<h4>Methods</h4>C57BL6 mice underwent 60 min of partial hepatic ischemia followed by 15 min-24 hrs of reperfusion. We collected blood and liver samples, isolated circulating microparticles, and determined protein and lipid content. To establish mechanism for microparticle production, we subjected murine primary hepatocytes to hypoxia-reoxygenation. Because microparticles express everted phosphatidylserine residues that are the target of annexin V, we analyzed the effects of an annexin V-homodimer (Diannexin or ASP8597) on post-ischemia microparticle production and function.<h4>Results</h4>Microparticles were detected in the circulation 15-30 min after post-ischemic reperfusion, and contained markers of SECs, platelets, natural killer T cells, and CD8+ cells; 4 hrs later, they contained markers of macrophages. Microparticles contained F2-isoprostanes, indicating oxidative damage to membrane lipids. Injection of mice with TNF-? increased microparticle formation, whereas Diannexin substantially reduced microparticle release and prevented IRI. Hypoxia-re-oxygenation generated microparticles from primary hepatocytes by processes that involved oxidative stress. Exposing cultured hepatocytes to preparations of microparticles isolated from the circulation during IRI caused injury involving mitochondrial membrane permeability transition. Microparticles also activated platelets and induced neutrophil migration in vitro. The inflammatory properties of microparticles involved activation of NF-?B and JNK, increased expression of E-selectin, P-selectin, ICAM-1 and VCAM-1. All these processes were blocked by coating microparticles with Diannexin.<h4>Conclusions</h4>Following hepatic IRI, microparticles circulate and can be taken up by hepatocytes, where they activate signaling pathways that mediate inflammation and hepatocyte injury. Diannexin prevents microparticle formation and subsequent inflammation.
Project description:The immunomodulatory activity of mesenchymal stem/stromal cells (MSCs) to suppress innate and adaptive immune responses offers a potent cell therapy for modulating inflammation and promoting tissue regeneration. However, the inflammatory cytokine milieu plays a critical role in stimulating MSC immunomodulatory activity. In particular, interferon-? (IFN-?)-induced expression of indoleamine 2,3-dioxygenase (IDO) is primarily responsible for MSC suppression of T-cell proliferation and activation. Although pretreatment with IFN-? is commonly used to prime MSCs for immunomodulatory activity prior to transplantation, the transient effects of pretreatment may limit the potential of MSCs to potently modulate immune responses. Therefore, the objective of this study was to investigate whether microparticle-mediated presentation of bioactive IFN-? within three-dimensional spheroidal MSC aggregates could precisely regulate and induce sustained immunomodulatory activity. Delivery of IFN-? via heparin-microparticles within MSC aggregates induced sustained IDO expression during 1 week of culture, whereas IDO expression by IFN-?-pretreated MSC spheroids rapidly decreased during 2 days. Furthermore, sustained IDO expression induced by IFN-?-loaded microparticles resulted in an increased and sustained suppression of T-cell activation and proliferation in MSC cocultures with CD3/CD28-activated peripheral blood mononuclear cells. The increased suppression of T cells by MSC spheroids containing IFN-?-loaded microparticles was dependent on induction of IDO and supported by affecting monocyte secretion from pro- to anti-inflammatory cytokines. Altogether, microparticle delivery of IFN-? within MSC spheroids provides a potent means of enhancing and sustaining immunomodulatory activity to control MSC immunomodulation after transplantation and thereby improve the efficacy of MSC-based therapies aimed at treating inflammatory and immune diseases. Stem Cells Translational Medicine 2017;6:223-237.
Project description:Septic shock is a severe disease state characterised by the body's life threatening response to infection. Complex interactions between endothelial cells and circulating monocytes are responsible for microvasculature dysfunction contributing to the pathogenesis of this syndrome. Here, we intended to determine whether microparticles derived from activated monocytes contribute towards inflammatory processes and notably vascular permeability. We found that endotoxin stimulation of human monocytes enhances the release of microparticles of varying phenotypes and mRNA contents. Elevated numbers of LPS-induced monocytic microparticles (mMP) expressed CD54 and contained higher levels of transcripts for pro-inflammatory cytokines such as TNF, IL-6 and IL-8. Using a prothrombin time assay, a greater reduction in plasma coagulation time was observed with LPS-induced mMP than with non-stimulated mMP. Co-incubation of mMP with the human brain endothelial cell line hCMEC/D3 triggered their time-dependent uptake and significantly enhanced endothelial microparticle release. Unexpectedly, mMP also modified signalling pathways by diminishing pSrc (tyr416) expression and promoted endothelial monolayer tightness, as demonstrated by endothelial impedance and permeability assays. Altogether, these data strongly suggest that LPS-induced mMP have contrasting effects on the intercellular communication network and display a dual potential: enhanced pro-inflammatory and procoagulant properties, together with protective function of the endothelium.
Project description:BACKGROUND AND OBJECTIVE:For many pathological states, microparticles are supposed to be one of the causes of hypercoagulation. Although there are some indirect data about microparticles participation in coagulation activation and propagation, the integral hemostasis test Thrombodynamics allows to measure micropaticles participation in these two coagulation phases directly. Demonstrates microparticles participation in coagulation activation by influence on the appearance of coagulation centres in the plasma volume and the rate of clot growth from the surface with immobilized tissue factor.Methods: Microparticles were obtained from platelets and erythrocytes by stimulation with thrombin receptor-activating peptide (SFLLRN) and calcium ionophore (A23187), respectively, from monocytes, endothelial HUVEC culture and monocytic THP cell culture by stimulation with lipopolysaccharides. Microparticles were counted by flow cytometry and titrated in microparticle-depleted normal plasma in the Thrombodynamics test. RESULTS:Monocyte microparticles induced the appearance of clotting centres through the TF pathway at concentrations approximately 100-fold lower than platelet and erythrocyte microparticles, which activated plasma by the contact pathway. For endothelial microparticles, both activation pathways were essential, and their activity was intermediate. Monocyte microparticles induced plasma clotting by the appearance of hundreds of clots with an extremely slow growth rate, while erythrocyte microparticles induced the appearance of a few clots with a growth rate similar to that from surface covered with high-density tissue factor. Patterns of clotting induced by platelet and endothelial microparticles were intermediate. Platelet, erythrocyte and endothelial microparticles impacts on the rate of clot growth from the surface with tissue factor did not differ significantly within the 0-200·103/ul range of microparticles concentrations. However, at concentrations greater than 500·103/ul, erythrocyte microparticles increased the stationary clot growth rate to significantly higher levels than do platelet microparticles or artificial phospholipid vesicles consisting of phosphatidylcholine and phosphatidylserine. CONCLUSION:Microparticles of different origins demonstrated qualitatively different characteristics related to coagulation activation and propagation.
Project description:Porous silicon (pSi) microparticles, in diverse sizes and shapes, can be functionalized to present pathogen-associated molecular patterns that activate dendritic cells. Intraperitoneal injection of MPL-adsorbed pSi microparticles, in contrast to free MPL, resulted in the induction of local inflammation, reflected in the recruitment of neutrophils, eosinophils and proinflammatory monocytes, and the depletion of resident macrophages and mast cells at the injection site. Injection of microparticle-bound MPL resulted in enhanced secretion of the T helper 1 associated cytokines IFN-? and TNF-? by peritoneal exudate and lymph node cells in response to secondary stimuli while decreasing the anti-inflammatory cytokine IL-10. MPL-pSi microparticles independently exhibited anti-tumor effects and enhanced tumor suppression by low dose doxorubicin nanoliposomes. Intravascular injection of the MPL-bound microparticles increased serum IL-1? levels, which was blocked by the IL-1 receptor antagonist Anakinra. The microparticles also potentiated tumor infiltration by dendritic cells, cytotoxic T lymphocytes, and F4/80+ macrophages, however, a specific reduction was observed in CD204+ macrophages.