Cardioprotection by remote ischemic preconditioning of the rat heart is mediated by extracellular vesicles

Remote ischemic preconditioning (RIPC) of the heart is exerted by brief ischemic insults affected on a remote organ or a remote area of the heart before a sustained cardiac ischemia. To date, little is known about the inter-organ transfer mechanisms of cardioprotection by RIPC. Exosomes and microvesicles/microparticles are vesicles of 30-100nm and 100-1000nm in diameter, respectively (collectively termed extracellular vesicles [EVs]). Their content of proteins, mRNAs and microRNAs, render EVs ideal conveyors of inter-organ communication. However, whether EVs are involved in RIPC, is unknown. Therefore, here we investigated whether (1) IPC induces release of EVs from the heart, and (2) EVs are necessary for cardioprotection by RIPC. Hearts of male Wistar rats were isolated and perfused in Langendorff mode. A group of donor hearts was exposed to 3x5-5min global ischemia and reperfusion (IPC) or 30min aerobic perfusion, while coronary perfusates were collected. Coronary perfusates of these hearts were given to another set of recipient isolated hearts. A group of recipient hearts received IPC effluent depleted of EVs by differential ultracentrifugation. Infarct size was determined after 30min global ischemia and 120min reperfusion. The presence or absence of EVs in perfusates was confirmed by dynamic light scattering, the EV marker HSP60 Western blot, and electron microscopy. IPC markedly increased EV release from the heart as assessed by HSP60. Administration of coronary perfusate from IPC donor hearts attenuated infarct size in non-preconditioned recipient hearts (12.9+/-1,6% vs. 25.0+/-2.7%), similarly to cardioprotection afforded by IPC (7.3+/-2.7% vs. 22.1+/-2.9%) on the donor hearts. Perfusates of IPC hearts depleted of EVs failed to exert cardioprotection in recipient hearts (22.0+/-2.3%). This is the first demonstration that EVs released from the heart after IPC are necessary for cardioprotection by RIPC, evidencing the importance of vesicular transfer mechanisms in remote cardioprotection

Extracelluláris vezikulák és hematológiai malignitásokban játszott szerepük

Absztrakt Extracelluláris vesiculák minden szervezetben képződnek. Három legintenzívebben vizsgált csoportjuk az apoptotikus testek, a microvesiculák és az exosomák. A sejtek közötti kommunikációban, immunreakciókban, angiogenezisben betöltött szerepük csak néhány az eddig megismertek közül. A fiziológiás folyamatok mellett sokféle betegségben leírták változásaikat; a patomechanizmusban betöltött szerepük mellett felvetődik potenciális használatuk biomarkerekként. A szerzők betekintést kívánnak nyújtani az extracelluláris vesiculák kutatásába, kiemelve azt a néhány tanulmányt, amely a hematológiai malignitásokra fókuszált. A microvesiculák és exosomák vérplazmában mért mennyisége, a terápia során megfigyelt minőségi változása miatt felmerült, hogy a diagnosztikában, prognosztikában, illetve a minimális residualis betegség monitorozásában is használhatók lehetnek. Akut myeloid leukaemiában a természetes ölősejtek aktivitásának szupresszálásában bizonyított a blasteredetű exosomák szerepe. Krónikus lymphoid leukaemiában a microvesiculák közreműködése valószínű a gyógyszer-rezisztencia kialakulásában is. Orv. Hetil., 2016, 157(35), 1379–1384. | Abstract Extracellular vesicles are produced in all organisms. The most intensively investigated categories of extracellular vesicles include apoptotic bodies, microvesicles and exosomes. Among a very wide range of areas, their role has been confirmed in intercellular communication, immune response and angiogenesis (in both physiological and pathological conditions). Their alterations suggest the potential use of them as biomarkers. In this paper the authors give an insight into the research of extracellular vesicles in general, and then focus on published findings in hematological malignancies. Quantitative and qualitative changes of microvesicles and exosomes may have value in diagnostics, prognostics and minimal residual disease monitoring of hematological malignancies. The function of extracellular vesicles in downregulation of natural killer cells’ activity has been demonstrated in acute myeloid leukemia. In chronic lymphocytic leukemia, microvesicles seem to play a role in drug resistance. Orv. Hetil., 2016, 157(35), 1379–1384

MMP Activity Detection in Zymograms

Matrix metalloproteinases (MMP) belong to a distinguished class of zinc-dependent endopeptidases. Zymography is a semi-quantitative tool for determining the activity of different MMP isoenzymes in a variety of biological samples. In substrate gel zymography, protein samples of different origin (tissue, cell lysates, plasma/serum, perfusates, other liquids) are separated in sodium dodecyl sulfate (SDS) polyacrylamide gels containing copolymerized substrate (gelatin, casein, elastin, etc.), and after incubation-enabling substrate cleavage by MMPs, MMP activities are detected after the gel staining as transparent bands against a dark-blue background. In situ zymography is a histological modification of substrate zymography in frozen sections, allowing detection of the localization of the MMP activities within the tissue. Here, we describe detailed experimental protocols of all abovementioned techniques and provide examples for several sample measurements

Antibiotic-induced release of small extracellular vesicles (exosomes) with surface-associated DNA

Recently, biological roles of extracellular vesicles (which include among others exosomes, microvesicles and apoptotic bodies) have attracted substantial attention in various fields of biomedicine. Here we investigated the impact of sustained exposure of cells to the fluoroquinolone antibiotic ciprofloxacin on the released extracellular vesicles. Ciprofloxacin is widely used in humans against bacterial infections as well as in cell cultures against Mycoplasma contamination. However, ciprofloxacin is an inducer of oxidative stress and mitochondrial dysfunction of mammalian cells. Unexpectedly, here we found that ciprofloxacin induced the release of both DNA (mitochondrial and chromosomal sequences) and DNA-binding proteins on the exofacial surfaces of small extracellular vesicles referred to in this paper as exosomes. Furthermore, a label-free optical biosensor analysis revealed DNA-dependent binding of exosomes to fibronectin. DNA release on the surface of exosomes was not affected any further by cellular activation or apoptosis induction. Our results reveal for the first time that prolonged low-dose ciprofloxacin exposure leads to the release of DNA associated with the external surface of exosomes

Adverse Effects on beta-Adrenergic Receptor Coupling: Ischemic Postconditioning Failed to Preserve Long-Term Cardiac Function

BACKGROUND: Ischemic preconditioning (IPC) and ischemic postconditioning (IPoC) are currently among the most efficient strategies protecting the heart against ischemia/reperfusion injury. However, the effect of IPC and IPoC on functional recovery following ischemia/reperfusion is less clear, particularly with regard to the specific receptor-mediated signaling of the postischemic heart. The current article examines the effect of IPC or IPoC on the regulation and coupling of beta-adrenergic receptors and their effects on postischemic left ventricular function. METHODS AND RESULTS: The beta-adrenergic signal transduction was analyzed in 3-month-old Wistar rats for each of the intervention strategies (Sham, ischemia/reperfusion, IPC, IPoC) immediately and 7 days after myocardial infarction. Directly after the infarction a cardioprotective potential was demonstrated for both IPC and IPoC: the infarct size was reduced, apoptosis and production of reactive oxygen species were lowered, and the myocardial tissue was preserved. Seven days after myocardial ischemia, only IPC hearts showed significant functional improvement. Along with a deterioration in fractional shortening, IPoC hearts no longer responded adequately to beta-adrenergic stimulation. The stabilization of beta-adrenergic receptor kinase-2 via increased phosphorylation of Mdm2 (an E3-ubiquitin ligase) was responsible for desensitization of beta-adrenergic receptors and identified as a characteristic specific to IPoC hearts. CONCLUSIONS: Immediately after myocardial infarction, rapid and transient activation of beta-adrenergic receptor kinase-2 may be an appropriate means to protect the injured heart from excessive stress. In the long term, however, induction and stabilization of beta-adrenergic receptor kinase-2, with the resultant loss of positive inotropic function, leads to the functional picture of heart failure

An implanted device enables in vivo monitoring of extracellular vesicle-mediated spread of pro-inflammatory mast cell response in mice

Abstract Mast cells have been shown to release extracellular vesicles (EVs) in vitro. However, EV-mediated mast cell communication in vivo remains unexplored. Primary mast cells from GFP-transgenic and wild type mice, were grown in the presence or absence of lipopolysaccharide (LPS), and the secreted EVs were separated from the conditioned media. Mast cell-derived EVs were next cultured with LPS-naïve mast cells, and the induction of TNF-α expression was monitored. In addition, primary mast cells were seeded in diffusion chambers that were implanted into the peritoneal cavities of mice. Diffusion chambers enabled the release of GFP+ mast cell-derived EVs in vivo into the peritoneal cavity. Peritoneal lavage cells were assessed for the uptake of GFP+ EVs and for TNF-α production. In vitro, LPS-stimulated mast cell-derived EVs were efficiently taken up by non-stimulated mast cells, and induced TNF-α expression in a TLR4, JNK and P38 MAPK dependent manner. In vivo, using implanted diffusion chambers, we confirmed the release and transmission of mast cell-derived EVs to other mast cells with subsequent induction of TNF-α expression. These data show an EV-mediated spreading of pro-inflammatory response between mast cells, and provide the first in vivo evidence for the biological role of mast cell-derived EVs

Radiolabeling of Extracellular Vesicles with (99m)Tc for Quantitative In Vivo Imaging Studies.

The biodistribution of extracellular vesicles (EVs) is a fundamental question in the field of circulating biomarkers, which has recently gained attention. Despite the capabilities of nuclear imaging methods, such as single-photon emission computed tomography, radioisotope labeling of EVs and the use of the aforementioned methods for in vivo studies hardly can be found in the literature. In this article, the authors describe a novel method for the radioisotope labeling of erythrocyte-derived EVs using the (99m)Tc-tricarbonyl complex. Moreover, the capability of the developed labeling method for in vivo biodistribution studies is demonstrated in a mouse model. The authors found that the intravenously administered (99m)Tc-labeled EVs mostly accumulated in the liver and spleen. The in vivo stability of the labeled EVs was assessed by the comparison of the obtained biodistribution of EVs with that of the free (99m)Tc-tricarbonyl. According to the authors' data, only a minor fraction of the radioactive label became detached from the EVs

Low-density lipoprotein mimics blood plasma-derived exosomes and microvesicles during isolation and detection

Circulating extracellular vesicles have emerged as potential new biomarkers in a wide variety of diseases. Despite the increasing interest, their isolation and purification from body fluids remains challenging. Here we studied human pre-prandial and 4 hours postprandial platelet-free blood plasma samples as well as human platelet concentrates. Using flow cytometry, we found that the majority of circulating particles within the size range of extracellular vesicles lacked common vesicular markers. We identified most of these particles as lipoproteins (predominantly low-density lipoprotein, LDL) which mimicked the characteristics of extracellular vesicles and also co-purified with them. Based on biophysical properties of LDL this finding was highly unexpected. Current state-of-the-art extracellular vesicle isolation and purification methods did not result in lipoprotein-free vesicle preparations from blood plasma or from platelet concentrates. Furthermore, transmission electron microscopy showed an association of LDL with isolated vesicles upon in vitro mixing. This is the first study to show co-purification and in vitro association of LDL with extracellular vesicles and its interference with vesicle analysis. Our data point to the importance of careful study design and data interpretation in studies using blood-derived extracellular vesicles with special focus on potentially co-purified LDL