Anat Aharon

Nitric oxide and superoxide dismutase modulate endothelial progenitor cell function in type 2 diabetes mellitus

BackgroundThe function of endothelial progenitor cells (EPCs), which are key cells in vascular repair, is impaired in diabetes mellitus. Nitric oxide (NO) and reactive oxygen species can regulate EPC functions. EPCs tolerate oxidative stress by upregulating superoxide dismutase (SOD), the enzyme that neutralizes superoxide anion (O2-). Therefore, we investigated the roles of NO and SOD in glucose-stressed EPCs.MethodsThe functions of circulating EPCs from patients with type 2 diabetes were compared to those from healthy individuals. Healthy EPCs were glucose-stressed, and then treated with insulin and/or SOD. We assessed O2- generation, NO production, SOD activity, and their ability to form colonies.ResultsEPCs from diabetic patients generated more O2-, had higher NAD(P)H oxidase and SOD activity, but lower NO bioavailability, and expressed higher mRNA and protein levels of p22-phox, and manganese SOD and copper/zinc SOD than those from the healthy individuals. Plasma glucose and HbA1c levels in the diabetic patients were correlated negatively with the NO production from their EPCs. SOD treatment of glucose-stressed EPCs attenuated O2- generation, restored NO production, and partially restored their ability to form colonies. Insulin treatment of glucose-stressed EPCs increased NO production, but did not change O2- generation and their ability to form colonies. However, their ability to produce NO and to form colonies was fully restored after combined SOD and insulin treatment.ConclusionOur data provide evidence that SOD may play an essential role in EPCs, and emphasize the important role of antioxidant therapy in type 2 diabetic patients.

Microparticles and pregnancy complications

Microparticles (MPs) are shed from cell membranes of a variety of cells, promote thrombus formation, mediate pro-inflammatory effects and may cause endothelial dysfunction. Normal pregnancy is characterized by increased levels of MPs compared to non-pregnant healthy women but the prevalence, cell origin and the role of MPs in pregnancy-related complications remain controversial. Normal pregnancy is an acquired hyper-coagulable state due to an increase in procoagulants and decrease in natural anticoagulants. Pregnancy-related complications such as preeclampsia, intrauterine fetal growth restriction (IUGR) and fetal loss are associated with placental dysfunction and may cause significant maternal and fetal morbidity and mortality. This article highlights the role of microparticles in maternal placental crosstalk and the interplay between microparticles, thrombosis and pregnancy complications.

Cryogenic Transmission Electron Microscopy Nanostructural Study of Shed Microparticles

Microparticles (MPs) are sub-micron membrane vesicles (100–1000 nm) shed from normal and pathologic cells due to stimulation or apoptosis. MPs can be found in the peripheral blood circulation of healthy individuals, whereas elevated concentrations are found in pregnancy and in a variety of diseases. Also, MPs participate in physiological processes, e.g., coagulation, inflammation, and angiogenesis. Since their clinical properties are important, we have developed a new methodology based on nano-imaging that provides significant new data on MPs nanostructure, their composition and function. We are among the first to characterize by direct-imaging cryogenic transmitting electron microscopy (cryo-TEM) the near-to-native nanostructure of MP systems isolated from different cell types and stimulation procedures. We found that there are no major differences between the MP systems we have studied, as most particles were spherical, with diameters from 200 to 400 nm. However, each MP population is very heterogeneous, showing diverse morphologies. We investigated by cryo-TEM the effects of standard techniques used to isolate and store MPs, and found that either high-g centrifugation of MPs for isolation purposes, or slow freezing to –80°C for storage introduce morphological artifacts, which can influence MP nanostructure, and thus affect the efficiency of these particles as future diagnostic tools.

Placenta-derived microparticles

Microparticles (MPs), sub-micron membrane vesicles, participate in the placental and maternal crosstalk in normal pregnancies as well as in gestational vascular complications (GVC). The article will review the effects of MPs on placental physiological processes, including hemostasis, trophoblast migration, invasion, placental vasculature and their involvement in pathologic states such as thrombosis, inflammation and endothelial dysfunction, resulting in GVC.

Disease dynamics in patients with acute myeloid leukemia: New biomarkers

Acute myeloid leukemia (AML) is characterized by rapid growth of leukemic blast cells. Extracellular vesicles (EVs), shedding from various cells, express antigens, reflecting their cellular origin. The current study was designed to explore the role of circulating EVs as potential biomarkers of AML activity and predictors of thrombogenicity in patients with this malignancy. Blood samples were collected from healthy controls and patients with newly diagnosed AML at three time points: diagnosis, nadir, and remission. EV concentration, cell origin, and expression of coagulation proteins were characterized using fluorescence-activated cell sorting. EV cytokine contents were evaluated by protein array. Procoagulant activity was assessed using Factor Xa chromogenic assay. Forty-two AML patients were enrolled in the study. Total EV numbers were higher in patients in first remission compared with controls, whereas blast EV counts were higher in patients at diagnosis compared with controls and patients in remission. Blast EV levels were significantly lower in patients who achieved remission and were alive at 3-year follow up compared with their succumbed counterparts. At all three time points, percentage of endothelial EVs was higher in patients compared with controls. EV procoagulant activity was elevated at diagnosis and in remission, and, unlike controls EVs, patients EVs increased endothelial cell thrombogenicity. EVs of AML patients express membrane proteins of blast cells and might serve as biomarkers of leukemia dynamics and presence of minimal residual disease. Increased levels of endothelial EVs and their procoagulant activity may indicate a vascular injury associated with a hypercoagulable state in AML.

A direct-imaging cryo-EM study of shedding extracellular vesicles from leukemic monocytes

The human leukemia monocytic cell line (THP-1) is known to shed extracellular vesicles (EVs) under various stimulations. We studied the effects of two types of common stimulation types, lipopolysaccharide (LPS) and starvation conditions by high resolution cryogenic electron microscopy, namely, cryo-SEM and cryo-TEM. Cryo-SEM data of cells undergoing EV blebbing and shedding is presented here for the first time. The high-resolution images show good agreement with models describing the membrane processes of shedding. Cells that underwent a 48-h starvation treatment exhibited differing morphological features, including shrunken nucleus and elongated membrane protrusions. LPS treated cells, however, showed extensive blebbing originating from the cell membrane, in good agreement with the sizes of EVs imaged by cryo-TEM. EVs isolated from both types of stimulations were measured by nanoparticle tracking analysis (NanoSight), by which LPS-EVs samples exhibited higher concentration and smaller mean diameter, as compared to starvation-EVs. Our results suggest a difference in the effects of the two stimulation types on the shedding process and possibly on the type of EVs shed. Our unique methodologies provide an important and innovative outlook of the shedding process and on its products, paving the way to further discoveries in this developing field of research, in which much is still unknown.

PO-46 - Influence of extracellular vesicles derived from AML patients on stem cells and their microenvironment

INTRODUCTIONnAcute myeloid leukemia (AML) is characterized by rapid growth of leukemic blast cells. Extracellular vesicles (EVs) are shed from normal and pathologic cells and express membrane proteins and antigens, reflecting their cellular origin.nnnAIMnTo explore whether bone marrow EVs of AML patients originate from blast cells and are capable of influencing hematopoietic stem cells (HSC) in a pseudo-natural microenvironment obtained by co-culture of HSC with mesenchymal stem cells (MSC).nnnMATERIALS AND METHODSnBone marrow (BM) samples were collected from healthy controls and patients with newly diagnosed AML at three time points: diagnosis, nadir and remission. EV concentration, cell origin and expression of coagulation proteins were characterized by FACS. Stem cells were obtained from Ficoll gradient of cord blood (CB) followed by CD34+ isolation. Cord blood stem cells with or without MSC were co-incubated with AML EVs. EV internalization was demonstrated by FACS-AMNIS and confocal microscopy. Mir-125b and -155 expressions in the cells were analyzed by RT-PCR.nnnRESULTSnAML patients were enrolled in the study. The total BM-EVs number was higher in patients at first remission compared to controls, while blast EV counts (labeled with anti-CD34, CD33, CD117) were higher in patients at diagnosis compared to controls and to patients in remission. Internalization of CD117+/CD33+ BM-EVs to cord blood stem cells in the presence or absence of MSC was evaluated by FACS-AMNIS. Confocal microscopy of CD33+ stained EVs strengthens the findings and shows presence of EVs even in the cytoplasm and the nucleus. Quantitative analysis of mir-125b and mir-155 expression in cord blood stem cells incubated with AML EVs revealed a clear tendency of increased expression in case of cell exposure to AML EVs in comparison to healthy control EVs. This tendency was emphasized in the presence of MSC.nnnCONCLUSIONSnEVs of AML patients are generated from blast cells. By internalization into naïve stem cells they can influence their differentiation. Moreover, the presence of mesenchymal stem cells is likely to be essential to the process.

Growth, developmental achievements and vaccines timeliness of undocumented migrant children from Eritrea compared with Israelis

Introduction Israel has absorbed >40,000 Eritrean undocumented migrants since 2007, while the majority live in the southern neighborhoods of Tel-Aviv. As non-citizens and citizens infants in Israel receive free preventive treatment at the mother and child health clinics (MCHC), this study aimed to compare development and growth achievements between children of Eritrean mothers (CE) to children of Israeli mothers (CI), and assess their compliance to routine follow-up and vaccination-timeliness. Methods This cohort study included all Israeli-born CE between 2009 and 2011, compared with a random sample of CI and treated at the same MCHC and followed-up to the age of 30-months. Dependent outcomes included anthropometric measurements, developmental achievements and adherence to immunization schedule. Results Of all 271 CE who were compared with 293 CI, no statistically significant differences were found in birth anthropometric measurements. Yet, CE had increased weight and length than CI after reaching one year of age (p<0.05). CE were more likely to fail in tests assessing fine-motor skills, linguistic and socio-emotional domains than CI, while no statistical difference was found in gross-motor achievements. At the end of follow-up, 203 (74.9%) of the CE and 271 (74.1%) of the CI completed the vaccination schedule, p = 0.9. Conclusion CE had greater anthropometric measurements than CI after one year of age, and showed higher impairments in fine motor, linguistic and socio-emotional domains. Adherence to vaccination was similar. The inequalities in child health should be responded in the MCTC, and Eritrean mothers should be trained with the current recommendations for child well-being.