Endothelium-derived microparticles impair endothelial function in vitro

Endothelial cell dysfunction (ECD) is emerging as the common denominator for diverse and highly prevalent cardiovascular diseases. Recently, an increased number of procoagulant circulating endothelial microparticles (EMPs) has been identified in patients with acute myocardial ischemia, preeclampsia, and diabetes, which suggests that these particles represent a surrogate marker of ECD. Our previous studies showed procoagulant potential of endothelial microparticles and mobilization of microparticles by PAI-1. The aim of this study was to test the effects of isolated EMPs on the vascular endothelium. EMPs impaired ACh-induced vasorelaxation and nitric oxide production by aortic rings obtained from Sprague-Dawley rats in a concentration-dependent manner. This effect was accompanied by increased superoxide production by aortic rings and cultured endothelial cells that were coincubated with EMPs and was inhibited by a SOD mimetic and blunted by an endothelial nitric oxide synthase inhibitor. Superoxide was also produced by isolated EMP. In addition, p22(phox) subunit of NADPH-oxidase was detected in EMP. Our data strongly suggest that circulating EMPs directly affect the endothelium and thus not only act as a marker for ECD but also aggravate preexisting ECD.     

Increased circulating endothelial microparticles in children with FMF

Objective: Endothelial microparticles (EMPs) are considered as markers of endothelial dysfunction. In this study, we aimed to examine whether there is endothelial dysfunction in children with familial Mediterranean fever (FMF), hypothesizing that endothelial dysfunction would be present especially with acute-phase response in the active period of the disease.

Methods: This cross-sectional study included 65 FMF patients (41 attack free, 24 attack period) and 35 healthy controls. Circulating EMPs, serum amyloid A (SAA), and other inflammation markers were measured in all groups. Circulating EMPs were measured using flow cytometry. Study groups were compared for circulating EMP and inflammatory markers. The relationship between EMPs and the activation of the disease was evaluated.

Results: The levels of CD144⁺ and CD146⁺ EMPs in the FMF attack period group were significantly higher than those of the control group (p?p?+ and CD146⁺ EMP were significantly correlated with CRP.

Conclusions: Our results suggest that endothelial damage is present especially in the active period of the disease in children with FMF. The endothelial dysfunction becomes an overt parallel with inflammation.     

Comparative proteomic analysis of PAI-1 and TNF-alpha-derived endothelial microparticles

Endothelium-derived microparticles (EMPs) are small vesicles released from endothelial cells in response to cell injury, apoptosis, or activation. Elevated concentrations of EMPs have been associated with many inflammatory and vascular diseases. EMPs also mediate long range signaling and alter downstream cell function. Unfortunately, the molecular and cellular basis of microparticle production and downstream cell function is poorly understood. We hypothesize that EMPs generated by different agonists will produce distinct populations of EMPs with unique protein compositions. To test this hypothesis, different EMP populations were generated from human umbilical vein endothelial cells by stimulation with plasminogen activator inhibitor type 1 (PAI-1) or tumor necrosis factor-alpha (TNF-alpha) and subjected to proteomic analysis by LC/MS. We identified 432 common proteins in all EMP populations studied. Also identified were 231 proteins unique to control EMPs, 104 proteins unique to PAI-1 EMPs and 70 proteins unique to TNF-alpha EMPs. Interestingly, variations in protein abundance were found among many of the common EMP proteins, suggesting that differences exist between EMPs on a relative scale. Finally, gene ontology (GO) and KEGG pathway analysis revealed many functional similarities and few differences between the EMP populations studied. In summary, our results clearly indicate that EMPs generated by PAI-1 and TNF-alpha produce EMPs with overlapping but distinct protein compositions. These observations provide fundamental insight into the mechanisms regulating the production of these particles and their physiological role in numerous diseases.     

Statin decreases endothelial microparticle release from human coronary artery endothelial cells: implication for the Rho-kinase pathway

OBJECTIVE: Elevated plasma levels of endothelial microparticles (EMPs) are associated with the presence of clinical atherosclerosis. Considering the anti-inflammatory properties of HMG-CoA reductase inhibitors on the endothelium, we studied the effect of fluvastatin on the release of EMPs in cultured human coronary artery endothelial cells (HCAEC). METHODS AND RESULTS: EMPs were generated in TNF-alpha-activated HCAECs. The absolute number of EMPs was enumerated using a novel two-color flow cytometric immunostaining technique with TruCount beads as an internal reference. EMPs are defined as EC membrane vesicles (1-2 microm in size) with a characteristic immunophenotype. The addition of fluvastatin to TNF-alpha-activated HCAECs significantly suppressed EMP release. Fluvastatin suppressed TNF-alpha-induced Rho activation.The Rho-kinase inhibitor, Y-27632, reproduced the effect of statin. CONCLUSION: EMP release from TNF-alpha-activated HCAECs is suppressed by fluvastatin. In addition, the Rho/Rho-kinase may play an important role in modulating EMP release.     

The Golgi-localized Arabidopsis endomembrane protein12 contains both endoplasmic reticulum export and Golgi retention signals at its C terminus

Endomembrane proteins (EMPs), belonging to the evolutionarily conserved transmembrane nine superfamily in yeast and mammalian cells, are characterized by the presence of a large lumenal N terminus, nine transmembrane domains, and a short cytoplasmic tail. The Arabidopsis thaliana genome contains 12 EMP members (EMP1 to EMP12), but little is known about their protein subcellular localization and function. Here, we studied the subcellular localization and targeting mechanism of EMP12 in Arabidopsis and demonstrated that (1) both endogenous EMP12 (detected by EMP12 antibodies) and green fluorescent protein (GFP)-EMP12 fusion localized to the Golgi apparatus in transgenic Arabidopsis plants; (2) GFP fusion at the C terminus of EMP12 caused mislocalization of EMP12-GFP to reach post-Golgi compartments and vacuoles for degradation in Arabidopsis cells; (3) the EMP12 cytoplasmic tail contained dual sorting signals (i.e., an endoplasmic reticulum export motif and a Golgi retention signal that interacted with COPII and COPI subunits, respectively); and (4) the Golgi retention motif of EMP12 retained several post-Golgi membrane proteins within the Golgi apparatus in gain-of-function analysis. These sorting signals are highly conserved in all plant EMP isoforms and, thus, likely represent a general mechanism for EMP targeting in plant cells.     

Age dependent association of endometrial polyps with increased risk of cancer involvement

BACKGROUND: Endometrial polyps (EMPs) are commonly encountered in routine surgical pathology practice, but opinions differ on whether they are intrinsically a marker for concurrent or subsequent malignancy. The objectives of the present study are 1) to investigate the age-group in which EMP are most commonly encountered 2) to document the age-group in which EMP are most commonly associated with malignancies 3) To investigate whether the age of diagnosis of the various carcinoma subtypes in EMPs is congruent with published data on similar malignancies arising in non-polypoid endometrium and 4) To investigate whether the histologic subtype distribution of malignancies associated with EMPs are similar or different from the distribution of malignancies arising from non-polypoid endometrium based on published data. PATIENTS AND METHODS: All cases of EMPs were retrieved from the files of Yale-New Haven Hospital for the period 1986-1995. The patients were divided into 5 age groups: Each group was further subclassified based on an association (or lack thereof) of EMPs with endometrial carcinoma. Chi-square test was used to compare the proportion of malignancy associated EMPs between the age groups. RESULTS: We identified 513 EMPs, of which 209 (41%) were from biopsy specimens and 304 (59%) from hysterectomy specimens. Sixty six (13%) of all EMPs were malignant. The 66 malignant EMPs included 58 endometrioid, 6 serous, 1 carcinosarcoma, and 1 clear cell carcinoma. In age group >35, only 1(2.5%) of 40 EMPs was associated with endometrial malignancy. In contrast, 37(32%) of 115 EMPs were associated with malignancy in the age group > 65. The frequency of malignant EMPs increased with age and reached statistical significance in the age group >65 (p < 0.001). The most common histologic type of malignancy was endometrioid adenocarcinoma. CONCLUSIONS: EMPs show statistically significant age dependent association with malignant tumor involvement. Careful search for malignancy, particularly in women with multiple risk factors is advised in daily practice. Additional studies are needed to address the histological features and immunohistochemical profiles in the context of association between endometrioid and high-grade endometrial carcinoma and endometrial polyps.     

Characterization of new multimeric erythropoietin receptor agonists

In addition to its natural ligand, the receptor for erythropoietin can be activated by small peptides known as erythropoietin mimetic peptides (EMPs). Although EMPs are less potent than the natural ligand, EMP dimers, consisting of two EMPs joined via a linker, have been shown to exhibit significantly improved activity compared to the corresponding monomers, with potency approaching that of the native hormone. In this study, we used a panel of novel EMP dimers to explore the effects of linker length and EMP attachment site on potency. The EC50 values obtained in an EPO-dependent proliferation assay indicated that, as has been shown with similar molecules, EMP dimerization can lead to increases in potency of more than 2 orders of magnitude. We found that both C-terminal and N-terminal attachment of the linker to EMP was tolerated, and that, with the exception of the shortest linker, all of the linker lengths tested provided a similar increase in potency. In follow-up work devised to explore the potential benefit of contacting additional cell surface EPO receptors, we designed a tetrameric template consisting of lysine-based dimers joined via commercial PEG linkers of various molecular weights. Evaluation of the resulting molecules indicated a clear effect of PEG linker size on activity, while the "dimer of dimer" with the shortest linker exhibited 10-fold lower potency than the corresponding dimer, the longest tetramer increased potency by fivefold. We discuss the implications of these results for the further development of EMP multimers.     

EMP‐induced BBB‐disruption enhances drug delivery to glioma and increases treatment efficacy in rats

Chemotherapy on gliomas is not satisfactorily efficient because the presence of blood‐brain barriers (BBB) leads to inadequate exposure of tumor cells to administered drugs. In order to facilitate chemotherapeutics to penetrate BBB and increase the treatment efficacy of gliomas, electromagnetic pulse (EMP) was applied and the 1‐(2‐Chlorethyl)‐cyclohexyl‐nitrosourea (CCNU) lomustine concentration in tumor tissue, tumor size, tumor apoptosis, and side effects were measured in glioma‐bearing rat model. The results showed that EMP exposure could enhance the delivery of CCNU to tumor tissue, facilitate tumor apoptosis, and inhibit tumor growth without obvious side effects. The data indicated that EMP‐induced BBB disruption could enhance delivery of CCNU to glioblastoma multiforme and increase treatment efficacy in glioma‐bearing rats. Bioelectromagnetics. 39:60–67, 2018. © 2017 Wiley Periodicals, Inc.     

Properties of avian, bovine and porcine erythrocyte membranes

Bovine, porcine and avian EMP were isolated and compared for some physical and chemical properties. Some differences in the compositions of three EMPs were observed. The avian EMP contained less carbohydrate than the bovine and porcine EMPs. Some differences in the monosaccharide distributions for the three preparations were revealed. The profiles obtained by SDS-polyacrylamide gel electrophoresis of the preparation indicated a complex (and different for each preparation) nature of the component polypeptides and glycopeptides.     

Conserved function of the lysine-based KXD/E motif in Golgi retention for endomembrane proteins among different organisms

We recently identified a new COPI-interacting KXD/E motif in the C-terminal cytosolic tail (CT) of Arabidopsis endomembrane protein 12 (AtEMP12) as being a crucial Golgi retention mechanism for AtEMP12. This KXD/E motif is conserved in CTs of all EMPs found in plants, yeast, and humans and is also present in hundreds of other membrane proteins. Here, by cloning selective EMP isoforms from plants, yeast, and mammals, we study the localizations of EMPs in different expression systems, since there are contradictory reports on the localizations of EMPs. We show that the N-terminal and C-terminal GFP-tagged EMP fusions are localized to Golgi and post-Golgi compartments, respectively, in plant, yeast, and mammalian cells. In vitro pull-down assay further proves the interaction of the KXD/E motif with COPI coatomer in yeast. COPI loss of function in yeast and plants causes mislocalization of EMPs or KXD/E motif–containing proteins to vacuole. Ultrastructural studies further show that RNA interference (RNAi) knockdown of coatomer expression in transgenic Arabidopsis plants causes severe morphological changes in the Golgi. Taken together, our results demonstrate that N-terminal GFP fusions reflect the real localization of EMPs, and KXD/E is a conserved motif in COPI interaction and Golgi retention in eukaryotes.     

Effects of High Intensity Training and High Volume Training on Endothelial Microparticles and Angiogenic Growth Factors

Aims

Endothelial microparticles (EMP) are complex vesicular structures shed from activated or apoptotic endothelial cells. As endurance exercise affects the endothelium, the objective of the study was to examine levels of EMP and angiogenic growth factors following different endurance exercise protocols.

Methods

12 subjects performed 3 different endurance exercise protocols: 1. High volume training (HVT; 130 min at 55% peak power output (PPO); 2. 4×4 min at 95% PPO; 3. 4×30 sec all-out. EMPs were quantified using flow cytometry after staining platelet-poor-plasma. Events positive for Annexin-V and CD31, and negative for CD42b, were classified as EMPs. Vascular endothelial growth factor (VEGF), migratory inhibiting factor (MIF) and hepatocyte growth factor (HGF) were determined by ELISA technique. For all these measurements venous blood samples were taken pre, 0′, 30′, 60′ and 180′ after each intervention. Furthermore, in vitro experiments were performed to explore the effect of collected sera on target endothelial functions and MP uptake capacities.

Results

VEGF and HGF significantly increased after HIT interventions. All three interventions caused a significant decrease in EMP levels post exercise compared to pre values. The sera taken after exercise increased the uptake of EMP in target endothelial cells compared to sera taken under resting conditions, which was shown to be phosphatidylserin-dependent. Increased EMP uptake was associated with an improved protection of target cells against apoptosis. Sera taken prior and after exercise promoted target endothelial cell migration, which was abrogated after inhibition of VEGF.

Conclusion

Physical exercise leads to decreased EMP levels and promotes a phosphatidylserin-dependent uptake of EMP into target endothelial cells, which is associated with a protection of target cells against apoptosis.     

Combined experimental/computational aspects for the molecular interaction of empagliflozin with bovine serum albumin: Quantification and application to human plasma

Empagliflozin (EMP) is an oral antihyperglycemic agent for type 2 diabetic patients. The molecular binding of EMP to bovine serum albumin (BSA) was elucidated by a combined experimental/computational approach to fulfil the pharmacokinetics and pharmacodynamics gaps of the cited drug for further development. Fluorescence, synchronous, and three-dimensional fluorescence spectroscopy verified that EMP quenched BSA native fluorescence through a dual static/dynamic mechanism that was further supported by Fӧrster resonance energy transfer and ultraviolet absorption spectroscopy. Fourier transform infrared spectroscopy revealed the conformational variations in BSA secondary structure induced by EMP. Thermodynamic properties of the BSA–EMP complex were also investigated, and the hydrophobic interactions' role in the binding process was demonstrated by the computed enthalpy (ΔH = 6.558 kJ mol⁻¹) and entropy (ΔS = 69.333 J mol⁻¹ K⁻¹). Gibbs free energy (ΔG) values were negative at three distinct temperatures, illuminating the spontaneity of this interaction. In addition, molecular docking studies depicted the optimal fitting of EMP to BSA on Site I (sub-domain IIA) through three hydrogen bonds. Additionally, and based on the quenching effect of EMP on BSA fluorescence, this study suggests a simple validated spectrofluorometric method for the quantitation of the studied drug in bulk form and human plasma samples with reasonable recoveries (96.99–103.10%).     

Endothelial microparticles reduce ICAM‐1 expression in a microRNA‐222‐dependent mechanism

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE−/− mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced.     

Effects of 900 MHz Radiofrequency Radiation on Skin Hydroxyproline Contents

The present study aimed to investigate the possible effect of pulse-modulated radiofrequency radiation (RFR) on rat skin hydroxyproline content, since skin is the first target of external electromagnetic fields. Skin hydroxyproline content was measured using liquid chromatography mass spectrometer method. Two months old male wistar rats were exposed to a 900 MHz pulse-modulated RFR at an average whole body specific absorption rate (SAR) of 1.35 W/kg for 20 min/day for 3 weeks. The radiofrequency (RF) signals were pulse modulated by rectangular pulses with a repetition frequency of 217 Hz and a duty cycle of 1:8 (pulse width 0.576 ms). A skin biopsy was taken at the upper part of the abdominal costa after the exposure. The data indicated that whole body exposure to a pulse-modulated RF radiation that is similar to that emitted by the global system for mobile communications (GSM) mobile phones caused a statistically significant increase in the skin hydroxyproline level (p = 0.049, Mann–Whitney U test). Under our experimental conditions, at a SAR less than the International Commission on Non-Ionizing Radiation Protection safety limit recommendation, there was evidence that GSM signals could alter hydroxyproline concentration in the rat skin.     

Endothelial microparticles act as novel diagnostic and therapeutic biomarkers of circulatory hypoxia‐related diseases: a literature review

Circulatory hypoxia‐related diseases (CHRDs), including acute coronary syndromes, stroke and organ transplantation, attract increased attention due to high morbidity and mortality. Mounting evidence shows that hypoxia‐induced oxidative stress, coagulation, inflammation and angiogenesis play extremely important roles in the physiological and pathological processes of CHRD‐related vascular endothelial injury. Interestingly, hypoxia, even hypoxia‐induced oxidative stress, coagulation and inflammation can all induce release of endothelial microparticles (EMPs). EMPs, shed from activated or apoptotic endothelial cells (ECs), reflect the degree of EC damage, and elevated EMP levels are found in several CHRDs. Furthermore, EMPs, which play an important role in cell‐to‐cell communication and function, have confirmed pro‐coagulant, proinflammatory, angiogenic and other functions, affecting pathological processes. These findings suggest that EMPs and CHRDs have a very close relationship, and EMPs may help to identify CHRD phenotypes and stratify the severity of disease, to improve risk stratification for developing CHRDs, to better define prophylactic strategies and to ameliorate prognostic characterization of patients with CHRDs. This review summarizes the known and potential roles of EMPs in the diagnosis, staging, treatment and clinical prognosis of CHRDs.     

Absorption of microwave radiation by the anesthetized rat: electromagnetic and thermal hotspots in body and tail

Anatomic variability in the deposition of radiofrequency electromagnetic energy in mammals has been well documented. A recent study [D'Andrea et al., 1985] reported specific absorption rate (SAR) hotspots in the brain, rectum and tail of rat carcasses exposed to 360- and to 2,450-MHz microwave radiation. Regions of intense energy absorption are generally thought to be of little consequence when predicting thermal effects of microwave irradiation because it is presumed that heat transfer via the circulatory system promptly redistributes localized heat to equilibrate tissue temperature within the body. Experiments on anesthetized, male Long-Evans rats (200-260 g) irradiated for 10 or 16 min with 2,450, 700, or 360 MHz radiation at SARs of 2 W/kg, 6 W/kg, or 10 W/kg indicated that postirradiation localized temperatures in regions previously shown to exhibit high SARs were appreciably above temperatures at body sites with lower SARs. The postirradiation temperatures in the rectum and tail were significantly higher in rats irradiated at 360 MHz and higher in the tail at 2,450 MHz than temperatures resulting from exposure to 700 MHz. This effect was found for whole-body-averaged SARs as low as 6 W/kg at 360 MHz and 10 W/kg at 2,450 MHz. In contrast, brain temperatures in the anesthetized rats were not different from those measured in the rest of the body following microwave exposure.     

Electromagnetic pulse reduces free radical generation in rat liver mitochondria in vitro

Abstract

Non-ionizing radiation electromagnetic pulse (EMP) is generally recorded to induce the generation of free radicals in vivo. Though mitochondria are the primary site to produce free radicals, a rare report is designed to directly investigate the EMP effects on free radical generation at mitochondrial level. Thus the present work was designed to study how EMP induces free radical generation in rat liver mitochondria in vitro using electron paramagnetic resonance technique. Surprisingly, our data suggest that EMP prevents free radical generation by activating antioxidant enzyme activity and reducing oxygen consumption and therefore free radical generation. Electron spin resonance measurements clearly demonstrate that disordering of mitochondrial lipid fluidity and membrane proteins mobility are the underlying contributors to this decreased oxygen consumption. Therefore, our results suggest that EMP might hold the potentiality to be developed as a non-invasive means to benefit certain diseases.     

Ectopic study of tissue-engineered bone complex with enamel matrix proteins, bone marrow stromal cells in porous calcium phosphate cement scaffolds, in nude mice

Objective: This study aimed to investigate the potential of enamel matrix proteins (EMPs) on promoting osteogenic differentiation of porcine bone marrow stromal cells (pBMSCs), as well as new bone formation capabilities, in a tissue‐engineered bone complex scaffold of EMPs, pBMSCs and porous calcium phosphate cement (CPC). Materials and methods: Effects of EMPs on pBMSCs in vitro was first determined by alkaline phosphatase (ALP) activity, von Kossa staining assay and mRNA expression of ALP, bone sialoprotein (BSP) and osteocalcin (OCN) genes. Next, an ectopic new bone formation test was performed in a nude mouse model with four groups: CPC scaffold alone; CPC scaffold + EMPs; CPC scaffold + pBMSCs; and CPC scaffold + EMPs + pBMSCs, for 2 or 4 weeks. Results: ALP activity, von Kossa assay and mRNA expressions of ALP, BSP and OCN genes were all significantly higher with 150 μg/ml EMP treatment in vitro. In nude mice, new bone formation was detected only in the CPC scaffold + EMPs + pBMSCs group at 2 weeks. At 4 weeks, in the tissue‐engineered construct there was significantly higher bone formation ability than other groups. Conclusions: EMPs promoted osteogenic differentiation of pBMSCs, and the tissue‐engineered complex of EMPs, pBMSCs and CPC scaffold may be a valuable alternative to be used in periodontal bone tissue engineering and regeneration.     

Kit ligand has a critical role in mouse yolk sac and aorta–gonad–mesonephros hematopoiesis

Few studies report on the in vivo requirement for hematopoietic niche factors in the mammalian embryo. Here, we comprehensively analyze the requirement for Kit ligand (Kitl) in the yolk sac and aorta–gonad–mesonephros (AGM) niche. In‐depth analysis of loss‐of‐function and transgenic reporter mouse models show that Kitl‐deficient embryos harbor decreased numbers of yolk sac erythro‐myeloid progenitor (EMP) cells, resulting from a proliferation defect following their initial emergence. This EMP defect causes a dramatic decrease in fetal liver erythroid cells prior to the onset of hematopoietic stem cell (HSC)‐derived erythropoiesis, and a reduction in tissue‐resident macrophages. Pre‐HSCs in the AGM require Kitl for survival and maturation, but not proliferation. Although Kitl is expressed widely in all embryonic hematopoietic niches, conditional deletion in endothelial cells recapitulates germline loss‐of‐function phenotypes in AGM and yolk sac, with phenotypic HSCs but not EMPs remaining dependent on endothelial Kitl upon migration to the fetal liver. In conclusion, our data establish Kitl as a critical regulator in the in vivoAGM and yolk sac endothelial niche.