Characterisation of adipocyte-derived extracellular vesicles released pre- and post-adipogenesis

Extracellular vesicles (EVs) are submicron vesicles released from many cell types, including adipocytes. EVs are implicated in the pathogenesis of obesity-driven cardiovascular disease, although the characteristics of adipocyte-derived EVs are not well described. We sought to define the characteristics of adipocyte-derived EVs before and after adipogenesis, hypothesising that adipogenesis would affect EV structure, molecular composition and function. Using 3T3-L1 cells, EVs were harvested at day 0 and day 15 of differentiation. EV and cell preparations were visualised by electron microscopy and EVs quantified by nanoparticle tracking analysis (NTA). EVs were then assessed for annexin V positivity using flow cytometry; lipid and phospholipid composition using 2D thin layer chromatography and gas chromatography; and vesicular protein content by an immuno-phenotyping assay. Pre-adipogenic cells are connected via a network of protrusions and EVs at both time points display classic EV morphology. EV concentration is elevated prior to adipogenesis, particularly in exosomes and small microvesicles. Parent cells contain higher proportions of phosphatidylserine (PS) and show higher annexin V binding. Both cells and EVs contain an increased proportion of arachidonic acid at day 0. PREF-1 was increased at day 0 whilst adiponectin was higher at day 15 indicating EV protein content reflects the stage of adipogenesis of the cell. Our data suggest that EV production is higher in cells before adipogenesis, particularly in vesicles <300 nm. Cells at this time point possess a greater proportion of PS (required for EV generation) whilst corresponding EVs are enriched in signalling fatty acids, such as arachidonic acid, and markers of adipogenesis, such as PREF-1 and PPARγ

Oral Protein Therapy for the Future - Transport of Glycolipid-Modified Proteins: Vision or Fiction?

The reliable and early diagnosis of common complex multifactorial diseases depends on the individual determination of all (or as many as possible) polymorphisms of each susceptibility gene together with amount and type of the corresponding gene products and their downstream effects, including the synthesis and flux of metabolites and regulation of signalling processes. In addition, this system's biology-driven personalized diagnosis must be accompanied by options for personalized reliable and early therapy. In the midterm, the direct substitution or inhibition of the proteins encoded by the corresponding defective gene products of the susceptibility genes exerting lower or higher activity by administration of the `normal' proteins or inhibitory antibodies, respectively, seems to be most promising. The critical hurdle of oral bioavailability as well as transport into the cytoplasm of the target cells, if required, could be overcome by therapeutic proteins with carboxy-terminal modification by glycosylphosphatidylinositol (GPI). This may be deduced from recent experiments with rat adipocytes. Here this membrane-anchoring glycolipid structure induces the sequential transport of proteins from special regions of the plasma membrane via the surface of intracellular lipid droplets to special membrane vesicles, which are finally released from the adipocytes together with the associated GPI proteins. It remains to be studied whether similar molecular mechanisms operate in intestinal epithelial cells and may enable the transport of GPI proteins from the intestinal lumen into the blood stream. If so, modification of proteins encoded by (combinations of) susceptibility genes with GPI could significantly facilitate the personalized therapy of common diseases on the basis of `inborn' safety, efficacy, rapid realization and oral application. Copyright (C) 2010 S. Karger AG, Base

Circulating microparticles: square the circle

Background: The present review summarizes current knowledge about microparticles (MPs) and provides a systematic overview of last 20 years of research on circulating MPs, with particular focus on their clinical relevance. Results: MPs are a heterogeneous population of cell-derived vesicles, with sizes ranging between 50 and 1000 nm. MPs are capable of transferring peptides, proteins, lipid components, microRNA, mRNA, and DNA from one cell to another without direct cell-to-cell contact. Growing evidence suggests that MPs present in peripheral blood and body fluids contribute to the development and progression of cancer, and are of pathophysiological relevance for autoimmune, inflammatory, infectious, cardiovascular, hematological, and other diseases. MPs have large diagnostic potential as biomarkers; however, due to current technological limitations in purification of MPs and an absence of standardized methods of MP detection, challenges remain in validating the potential of MPs as a non-invasive and early diagnostic platform. Conclusions: Improvements in the effective deciphering of MP molecular signatures will be critical not only for diagnostics, but also for the evaluation of treatment regimens and predicting disease outcomes

Platelet-derived exosomes induce endothelial cell apoptosis through peroxynitrite generation: experimental evidence for a novel mechanism of septic vascular dysfunction

Abstract\ud \ud \ud \ud Introduction\ud \ud Several studies link hematological dysfunction to severity of sepsis. Previously we showed that platelet-derived microparticles from septic patients induce vascular cell apoptosis through the NADPH oxidase-dependent release of superoxide. We sought to further characterize the microparticle-dependent vascular injury pathway.\ud \ud \ud \ud Methods\ud \ud During septic shock there is increased generation of thrombin, TNF-α and nitric oxide (NO). Human platelets were exposed for 1 hour to the NO donor diethylamine-NONOate (0.5 μM), lipopolysaccharide (LPS; 100 ng/ml), TNF-α (40 ng/ml), or thrombin (5 IU/ml). Microparticles were recovered through filtration and ultracentrifugation and analyzed by electron microscopy, flow cytometry or Western blotting for protein identification. Redox activity was characterized by lucigenin (5 μM) or coelenterazine (5 μM) luminescence and by 4,5-diaminofluorescein (10 mM) and 2',7'-dichlorofluorescein (10 mM) fluorescence. Endothelial cell apoptosis was detected by phosphatidylserine exposure and by measurement of caspase-3 activity with an enzyme-linked immunoassay.\ud \ud \ud \ud Results\ud \ud Size, morphology, high exposure of the tetraspanins CD9, CD63, and CD81, together with low phosphatidylserine, showed that platelets exposed to NONOate and LPS, but not to TNF-α or thrombin, generate microparticles similar to those recovered from septic patients, and characterize them as exosomes. Luminescence and fluorescence studies, and the use of specific inhibitors, revealed concomitant superoxide and NO generation. Western blots showed the presence of NO synthase II (but not isoforms I or III) and of the NADPH oxidase subunits p22phox, protein disulfide isomerase and Nox. Endothelial cells exposed to the exosomes underwent apoptosis and caspase-3 activation, which were inhibited by NO synthase inhibitors or by a superoxide dismutase mimetic and totally blocked by urate (1 mM), suggesting a role for the peroxynitrite radical. None of these redox properties and proapoptotic effects was evident in microparticles recovered from platelets exposed to thrombin or TNF-α.\ud \ud \ud \ud Conclusion\ud \ud We showed that, in sepsis, NO and bacterial elements are responsible for type-specific platelet-derived exosome generation. Those exosomes have an active role in vascular signaling as redox-active particles that can induce endothelial cell caspase-3 activation and apoptosis by generating superoxide, NO and peroxynitrite. Thus, exosomes must be considered for further developments in understanding and treating vascular dysfunction in sepsis.LRL and MJ have research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP. MJ received a research grant from Sociedade Beneficente Israelita-Brasileira Hospital Albert Einstein.LRL and MJ have research grants from Fundação de Amparo a Pesquisa do Estado de São Paulo – FAPESP. MJ received a research grant from Sociedade Beneficente IsraelitaBrasileira Hospital Albert Einstein

Nitrite-derived nitric oxide reduces hypoxia-inducible factor 1α-mediated extracellular vesicle production by endothelial cells

Introduction Extracellular vesicles (EVs) are small, spherical particles enclosed by a phospholipid bilayer (∼30–1000 nm) released from multiple cell types, and have been shown to have pathophysiological roles in a plethora of disease states. The transcription factor hypoxia-inducible factor-1 (HIF-1) allows for adaptation of cellular physiology in hypoxia and may permit the enhanced release of EVs under such conditions. Nitric oxide (NO) plays a pivotal role in vascular homeostasis, and can modulate the cellular response to hypoxia by preventing HIF-1 accumulation. We aimed to selectively target HIF-1 via sodium nitrite (NaNO2) addition, and examine the effect on endothelial EV, size, concentration and function, and delineate the role of HIF-1 in EV biogenesis. Methods Endothelial (HECV) cells were exposed to hypoxic conditions (1% O2, 24 h) and compared to endothelial cells exposed to normoxia (21% O2) with and without the presence of sodium nitrite (NaNO2) (30 μM). Allopurinol (100 μM), an inhibitor of xanthine oxidoreductase, was added both alone and in combination with NaNO2 to cells exposed to hypoxia. EV and cell preparations were quantified by nanoparticle tracking analysis and confirmed by electron microscopy. Western blotting and siRNA were used to confirm the role of HIF-1α and HIF-2α in EV biogenesis. Flow cytometry and time-resolved fluorescence were used to assess the surface and intravesicular protein content. Results Endothelial (HECV) cells exposed to hypoxia (1% O2) produced higher levels of EVs compared to cells exposed to normoxia. This increase was confirmed using the hypoxia-mimetic agent desferrioxamine. Treatment of cells with sodium nitrite (NaNO2) reduced the hypoxic enhancement of EV production. Treatment of cells with the xanthine oxidoreductase inhibitor allopurinol, in addition to NaNO2 attenuated the NaNO2-attributed suppression of hypoxia-mediated EV release. Transfection of cells with HIF-1α siRNA, but not HIF-2α siRNA, prior to hypoxic exposure prevented the enhancement of EV release. Conclusion These data provide evidence that hypoxia enhances the release of EVs in endothelial cells, and that this is mediated by HIF-1α, but not HIF-2α. Furthermore, the reduction of NO2− to NO via xanthine oxidoreductase during hypoxia appears to inhibit HIF-1α-mediated EV production

Exosomes from patients with septic shock convey miRNAs related to inflammation and cell cycle regulation: new signaling pathways in sepsis?

Background: Exosomes isolated from plasma of patients with sepsis may induce vascular apoptosis and myocardial dysfunction by mechanisms related to inflammation and oxidative stress. Despite previous studies demonstrating that these vesicles contain genetic material related to cellular communication, their molecular cargo during sepsis is relatively unknown. In this study, we evaluated the presence of microRNAs (miRNAs) and messenger RNAs (mRNAs) related to inflammatory response and redox metabolism in exosomes of patients with septic shock. Methods: Blood samples were collected from 24 patients with septic shock at ICU admission and after 7 days of treatment. Twelve healthy volunteers were used as control subjects. Exosomes were isolated by ultracentrifugation, and their miRNA and mRNA content was evaluated by qRT-PCR array. Results: As compared with healthy volunteers, exosomes from patients with sepsis had significant changes in 65 exosomal miRNAs. Twenty-eight miRNAs were differentially expressed, both at enrollment and after 7 days, with similar kinetics (18 miRNAs upregulated and 10 downregulated). At enrollment, 35 differentially expressed miRNAs clustered patients with sepsis according to survival. The pathways enriched by the miRNAs of patients with sepsis compared with control subjects were related mostly to inflammatory response. The comparison of miRNAs from patients with sepsis according to hospital survival demonstrated pathways related mostly to cell cycle regulation. At enrollment, sepsis was associated with significant increases in the expression of mRNAs related to redox metabolism (myeloperoxidase, 64-foldPRDX3, 2.6-foldSOD2, 2.2-fold) and redox-responsive genes (FOXM1, 21-foldSELS, 16-foldGLRX2, 3.4-fold). The expression of myeloperoxidase mRNA remained elevated after 7 days (65-fold). Conclusions: Exosomes from patients with septic shock convey miRNAs and mRNAs related to pathogenic pathways, including inflammatory response, oxidative stress, and cell cycle regulation. Exosomes may represent a novel mechanism for intercellular communication during sepsis.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Research and Education Institute, Hospital Sirio-LibanesHosp Sirio Libanes, Res & Educ Inst, Rua Prof Daher Cutait 69, BR-01539001 Sao Paulo, SP, BrazilUniv Sao Paulo, Sao Paulo State Canc Inst, Sao Paulo, BrazilHosp Serv Publ Estadual Sao Paulo, Sao Paulo, BrazilUniv Fed Minas Gerais, Inst Ciencias Biol, Morphol Dept, Belo Horizonte, MG, BrazilUniv Sao Paulo, Sch Med, Heart Inst, Lab Immunol, Sao Paulo, BrazilUniv Estadual Paraiba, Ctr Ciencias & Tecnol, Campina Grande, BrazilLudwig Inst Canc Res, Sao Paulo, BrazilAC Camargo Canc Ctr, Int Res Ctr, Sao Paulo, BrazilUniv Sao Paulo, Sch Med, Div Clin Immunol & Allergy, Sao Paulo, BrazilUniv Fed Sao Paulo, Sao Paulo, BrazilUniv Sao Paulo, Emergency Med, Sao Paulo, BrazilUniv Fed Sao Paulo, Sao Paulo, BrazilFAPESP: 10/52554-1Web of Scienc