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N2O is currently the third most important anthropogenic greenhouse gas in terms of radiative forcing and its atmospheric mole fraction is rising steadily. To quantify the growth rate and its causes over the past decades, we performed a multi-site reconstruction of the atmospheric N2O mole fraction and isotopic composition using new and previously published firn air data collected from Greenland and Antarctica in combination with a firn diffusion and densification model. The multi-site reconstruction showed that while the global mean N2O mole fraction increased from (290 ± 1) nmol mol−1 in 1940 to (322 ± 1) nmol mol−1 in 2008, the isotopic composition of atmospheric N2O decreased by (−2.2 ± 0.2) ‰ for δ15Nav, (−1.0 ± 0.3) ‰ for δ18O, (−1.3 ± 0.6) ‰ for δ15Nα, and (−2.8 ± 0.6) ‰ for δ15Nβ over the same period. The detailed temporal evolution of the mole fraction and isotopic composition derived from the firn air model was then used in a two-box atmospheric model (comprising a stratospheric box and a tropospheric box) to infer changes in the isotopic source signature over time. The precise value of the source strength depends on the choice of the N2O lifetime, which we choose to fix at 123 years. The average isotopic composition over the investigated period is δ15Nav = (−7.6 ± 0.8) ‰ (vs. air-N2), δ18O = (32.2 ± 0.2) ‰ (vs. Vienna Standard Mean Ocean Water – VSMOW) for δ18O, δ15Nα = (−3.0 ± 1.9) ‰ and δ15Nβ = (−11.7 ± 2.3) ‰. δ15Nav, and δ15Nβ show some temporal variability, while for the other signatures the error bars of the reconstruction are too large to retrieve reliable temporal changes. Possible processes that may explain trends in 15N are discussed. The 15N site preference ( = δ15Nα − δ15Nβ) provides evidence of a shift in emissions from denitrification to nitrification, although the uncertainty envelopes are large.

We thoroughly characterized a system for thermal separation of organic carbon (OC) and elemental carbon (EC) for subsequent radiocarbon analysis. Different organic compounds as well as ambient aerosol filter samples were introduced into an oven system and combusted to CO2 in pure O2. The main objective was to test which combustion times and temperatures are best suited to separate OC and EC. The final separation step for OC was combustion at 360 °C for 15 min. Combustion at this temperature proved enough to remove several organic test substances from the filter (including high molecular weight humic acid) but did not remove substantial amounts of EC. For isolation of EC, OC first needs to be completely removed from the filter. This was achieved by water extraction of the filter, followed by combustion of the water insoluble OC at 360 °C and combustion at an intermediate temperature step of 2 min at 450 °C. This last step removed the most refractory OC together with some EC. Finally, the remaining EC was combusted to CO2 at 650 °C. The recovery of black carbon after the intermediate 450 °C step was approximately 80%. Several tests provided strong evidence that OC was removed efficiently during the intermediate temperature step: (i) brown carbon, indicative of refractory OC, was removed; (ii) the fraction modern of EC did not decrease significantly if the temperature of the intermediate step was further increased. Based on tests with various organic compounds, we estimated that charred organic carbon could contribute 4–8% to an elemental carbon sample that was isolated according to our method.

Air bubbles in ice core samples represent the only opportunity to study the mixing ratio and isotopic variability of palaeoatmospheric CH4 and N2O. The highest possible precision in isotope measurements is required to maximize the resolving power for CH4 and N2O sink and source reconstructions. We present a new setup to measure δ13C-CH4, δ15N-N2O and δ18O-N2O isotope ratios in one ice core sample and with one single IRMS instrument, with a precision of 0.09, 0.6 and 0.7‰, respectively, as determined on 0.6–1.6 nmol CH4 and 0.25–0.6 nmol N2O. The isotope ratios are referenced to the VPDB scale (δ13C-CH4), the N2-air scale (δ15N-N2O) and the VSMOW scale (δ18O-N2O). Ice core samples of 200–500 g are melted while the air is constantly extracted to minimize gas dissolution. A helium carrier gas flow transports the sample through the analytical system. We introduce a new gold catalyst to oxidize CO to CO2 in the air sample. CH4 and N2O are then separated from N2, O2, Ar and CO2 before they get pre-concentrated and separated by gas chromatography. A combustion unit is required for δ13C-CH4 analysis, which is equipped with a constant oxygen supply as well as a post-combustion trap and a post-combustion GC column (GC-C-GC-IRMS). The post-combustion trap and the second GC column in the GC-C-GC-IRMS combination prevent Kr and N2O interferences during the isotopic analysis of CH4-derived CO2. These steps increase the time for δ13C-CH4 measurements, which is used to measure δ15N-N2O and δ18O-N2O first and then δ13C-CH4. The analytical time is adjusted to ensure stable conditions in the ion source before each sample gas enters the IRMS, thereby improving the precision achieved for measurements of CH4 and N2O on the same IRMS. The precision of our measurements is comparable to or better than that of recently published systems. Our setup is calibrated by analysing multiple reference gases that were injected over bubble-free ice samples. We show that our measurements of δ13C-CH4 in ice core samples are generally in good agreement with previously published data after the latter have been corrected for krypton interferences.

Stable carbon isotope analysis of methane (δ13C of CH4) on atmospheric samples is one key method to constrain the current and past atmospheric CH4 budget. A frequently applied measurement technique is gas chromatography (GC) isotope ratio mass spectrometry (IRMS) coupled to a combustion-preconcentration unit. This report shows that the atmospheric trace gas krypton (Kr) can severely interfere during the mass spectrometric measurement, leading to significant biases in δ13C of CH4, if krypton is not sufficiently separated during the analysis. According to our experiments, the krypton interference is likely composed of two individual effects, with the lateral tailing of the doubly charged 86Kr peak affecting the neighbouring m/z 44 and partially the m/z 45 Faraday cups. Additionally, a broad signal affecting m/z 45 and especially m/z 46 is assumed to result from scattered ions of singly charged krypton. The introduced bias in the measured isotope ratios is dependent on the chromatographic separation, the krypton-to-CH4 mixing ratio in the sample, the focusing of the mass spectrometer as well as the detector configuration and can amount to up to several per mil in δ13C. Apart from technical solutions to avoid this interference, we present correction routines to a posteriori remove the bias.

Ongoing research is actively exploring the use of immune checkpoint inhibitors to treat solid tumors by inhibiting the PD-1/PD-L1 axis and reactivating the function of cytotoxic T effector cells. Many types of solid tumors, however, are characterized by a dense and stiff stroma and are difficult to treat. Mechanotherapeutics have formed a recent class of drugs that aim to restore biomechanical abnormalities of the tumor microenvironment, related to increased stiffness and hypo-perfusion. Here, we have developed a polymeric formulation containing pirfenidone, which has been successful in restoring the tumor microenvironment in breast tumors and sarcomas. We found that the micellar formulation can induce similar mechanotherapeutic effects to mouse models of 4T1 and E0771 triple negative breast tumors and MCA205 fibrosarcoma tumors but with a dose 100-fold lower than that of the free pirfenidone. Importantly, a combination of pirfenidone-loaded micelles with immune checkpoint inhibition significantly delayed primary tumor growth, leading to a significant improvement in overall survival and in a complete cure for the E0771 tumor model. Furthermore, the combination treatment increased CD4 + and CD8 + T cell infiltration and suppressed myeloid-derived suppressor cells, creating favorable immunostimulatory conditions, which led to immunological memory. Ultrasound shear wave elastography (SWE) was able to monitor changes in tumor stiffness during treatment, suggesting optimal treatment conditions. Micellar encapsulation is a promising strategy for mechanotherapeutics, and imaging methods, such as SWE, can assist their clinical translation.

A recent U.S. Food and Drug Administration report presented the currently available scientific information related to biological response to metal implants. In this work, a multilevel approach was employed to assess the implant-induced and biocorrosion-related inflammation in the adjacent vascular tissue using a mouse stent implantation model. The implications of biocorrosion on peri-implant tissue were assessed at the macroscopic level via in vivo imaging and histomorphology. Elevated matrix metalloproteinase activity, colocalized with the site of implantation, and histological staining indicated that stent surface condition and implantation time affect the inflammatory response and subsequent formation and extent of neointima. Hematological measurements also demonstrated that accumulated metal particle contamination in blood samples from corroded-stetted mice causes a stronger immune response. At the cellular level, the stent-induced alterations in the nanostructure, cytoskeleton, and mechanical properties of circulating lymphocytes were investigated. It was found that cells from corroded-stented samples exhibited higher stiffness, in terms of Young's modulus values, compared to noncorroded and sham-stented samples. Nanomechanical modifications were also accompanied by cellular remodeling, through alterations in cell morphology and stress (F-actin) fiber characteristics. Our analysis indicates that surface wear and elevated metal particle contamination, prompted by corroded stents, may contribute to the inflammatory response and the multifactorial process of in-stent restenosis. The results also suggest that circulating lymphocytes could be a novel nanomechanical biomarker for peri-implant tissue inflammation and possibly the early stage of in-stent restenosis. Large-scale studies are warranted to further investigate these findings.

Skeletal muscle growth and maintenance depend on two tightly regulated processes, myogenesis and muscle regeneration. Both processes involve a series of crucial regulatory molecules including muscle-specific microRNAs, known as myomiRs. We recently showed that four myomiRs, miR-1, miR-133a, miR-133b, and miR-206, are encapsulated within muscle-derived exosomes and participate in local skeletal muscle communication. Although these four myomiRs have been extensively studied for their function in muscles, no information exists regarding their endogenous and exosomal levels across age. Here we aimed to identify any age-related changes in the endogenous and muscle-derived exosomal myomiR levels during acute skeletal muscle growth. The four endogenous and muscle-derived myomiRs were investigated in five skeletal muscles (extensor digitorum longus, soleus, tibialis anterior, gastrocnemius, and quadriceps) of 2-week-1-year-old wild-type male mice. The expression of miR-1, miR-133a, and miR-133b was found to increase rapidly until adolescence in all skeletal muscles, whereas during adulthood it remained relatively stable. By contrast, endogenous miR-206 levels were observed to decrease with age in all muscles, except for soleus. Differential expression of the four myomiRs is also inversely reflected on the production of two protein targets; serum response factor and connexin 43. Muscle-derived exosomal miR-1, miR-133a, and miR-133b levels were found to increase until the early adolescence, before reaching a plateau phase. Soleus was found to be the only skeletal muscle to release exosomes enriched in miR-206. In this study, we showed for the first time an in-depth longitudinal analysis of the endogenous and exosomal levels of the four myomiRs during skeletal muscle development. We showed that the endogenous expression and extracellular secretion of the four myomiRs are associated to the function and size of skeletal muscles as the mice age. Overall, our findings provide new insights for the myomiRs' significant role in the first year of life in mice., Competing Interests: MP was employed by the company Theramir Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mytidou, Koutsoulidou, Zachariou, Prokopi, Kapnisis, Spyrou, Anayiotos and Phylactou.)

Myotonic dystrophy type 1 (DM1) is the most common adult-onset muscular dystrophy, primarily characterized by muscle wasting and weakness. Many biomarkers already exist in the rapidly developing biomarker research field that aim to improve patients' care. Limited work, however, has been performed on rare diseases, including DM1. We have previously shown that specific microRNAs (miRNAs) can be used as potential biomarkers for DM1 progression. In this report, we aimed to identify novel serum-based biomarkers for DM1 through high-throughput next-generation sequencing. A number of miRNAs were identified that are able to distinguish DM1 patients from healthy individuals. Two miRNAs were selected, and their association with the disease was validated in a larger panel of patients. Further investigation of miR-223-3p, miR-24-3p, and the four previously identified miRNAs, miR-1-3p, miR-133a-3p, miR-133b-3p, and miR-206-3p, showed elevated levels in a DM1 mouse model for all six miRNAs circulating in the serum compared to healthy controls. Importantly, the levels of miR-223-3p, but not the other five miRNAs, were found to be significantly downregulated in five skeletal muscles and heart tissues of DM1 mice compared to controls. This result provides significant evidence for its involvement in disease manifestation., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Exosomes are extracellular vesicles that are released from most cell types encapsulating specific molecular cargo. Exosomes serve as mediators of cell-to-cell and tissue-to-tissue communications under normal and pathological conditions. It has been shown that exosomes carrying muscle-specific miRNAs, myomiRs, are secreted from skeletal muscle cells in vitro and are elevated in the blood of muscle disease patients. The aim of this study was to investigate the secretion of exosomes encapsulating the four myomiRs from skeletal muscle tissues and to assess their role in inter-tissue communication between neighboring skeletal muscles in vivo. We demonstrate, for the first time, that isolated, intact skeletal muscle tissues secrete exosomes encapsulating the four myomiRs, miR-1, miR-133a, miR-133b, and miR-206. Notably, we show that the sorting of the four myomiRs within exosomes varies between skeletal muscles of different muscle fiber-type composition. miR-133a and miR-133b downregulation in TA muscles caused a reduction of their levels in neighboring skeletal muscles and in serum exosomes. In conclusion, our results reveal that skeletal muscle-derived exosomes encapsulate the four myomiRs, some of which enter the blood, while a portion is used for the local communication between proximal muscle tissues. These findings provide important evidence regarding novel pathways implicated in skeletal muscle function., (© 2021 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Treatment of breast cancer underwent extensive progress in recent years with molecularly targeted therapies. However, non-specific pharmaceutical approaches (chemotherapy) persist, inducing severe side-effects. Phytochemicals provide a promising alternative for breast cancer prevention and treatment. Specifically, resveratrol (res) is a plant-derived polyphenolic phytoalexin with potent biological activity but displays poor water solubility, limiting its clinical use. Here we have developed a strategy for delivering res using a newly synthesized nano-carrier with the potential for both diagnosis and treatment. Methods: Res-loaded nanoparticles were synthesized by the emulsion method using Pluronic F127 block copolymer and Vitamin E-TPGS. Nanoparticle characterization was performed by SEM and tunable resistive pulse sensing. Encapsulation Efficiency (EE%) and Drug Loading (DL%) content were determined by analysis of the supernatant during synthesis. Nanoparticle uptake kinetics in breast cancer cell lines MCF-7 and MDA-MB-231 as well as in MCF-10A breast epithelial cells were evaluated by flow cytometry and the effects of res on cell viability via MTT assay. Results: Res-loaded nanoparticles with spherical shape and a dominant size of 179±22 nm were produced. Res was loaded with high EE of 73±0.9% and DL content of 6.2±0.1%. Flow cytometry revealed higher uptake efficiency in breast cancer cells compared to the control. An MTT assay showed that res-loaded nanoparticles reduced the viability of breast cancer cells with no effect on the control cells. Conclusions: These results demonstrate that the newly synthesized nanoparticle is a good model for the encapsulation of hydrophobic drugs. Additionally, the nanoparticle delivers a natural compound and is highly effective and selective against breast cancer cells rendering this type of nanoparticle an excellent candidate for diagnosis and therapy of difficult to treat mammary malignancies., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Despite their wide clinical usage, stent functionality may be compromised by complications at the site of implantation, including early/late stent thrombosis and occlusion. Although several studies have described the effect of fluid-structure interaction on local haemodynamics, there is yet limited information on the effect of the stent presence on specific hemorheological parameters. The current work investigates the red blood cell (RBC) mechanical behavior and physiological changes as a result of flow through stented vessels. Blood samples from healthy volunteers were prepared as RBC suspensions in plasma and in phosphate buffer saline at 45% haematocrit. Self-expanding nitinol stents were inserted in clear perfluoroalkoxy alkane tubing which was connected to a syringe, and integrated in a syringe pump. The samples were tested at flow rates of 17.5, 35 and 70 ml/min, and control tests were performed in non-stented vessels. For each flow rate, the sample viscosity, RBC aggregation and deformability, and RBC lysis were estimated. The results indicate that the presence of a stent in a vessel has an influence on the hemorheological characteristics of blood. The viscosity of all samples increases slightly with the increase of the flow rate and exposure. RBC aggregation and elongation index (EI) decrease as the flow rate and exposure increases. RBC lysis for the extreme cases is evident. The results indicate that the stresses developed in the stent area for the extreme conditions could be sufficiently high to influence the integrity of the RBC membrane.

Myotonic dystrophy type 1 (DM1) is the most common form of adult-onset muscular dystrophy, which is characterised by progressive muscle wasting and the discovery of reliable blood-based biomarkers could be useful for the disease progress monitoring. There have been some reports showing that the presence of specific miRNAs in blood correlates with DM1. In one of these, our group identified four muscle-specific miRNAs, miR-1, miR-133a, miR-133b and miR-206, which correlated with the progression of muscle wasting observed in DM1 patients. The levels of the four muscle-specific miRNAs were elevated in the serum of DM1 patients compared to healthy participants and were also elevated in the serum of progressive muscle wasting DM1 patients compared to disease-stable DM1 patients. The aim of this work was to characterise the ontology of these four muscle-specific miRNAs in the blood circulation of DM1 patients. Here we show that the four muscle-specific miRNAs are encapsulated within exosomes isolated from DM1 patients. Our results show for the first time, the presence of miRNAs encapsulated within exosomes in blood circulation of DM1 patients. More interestingly, the levels of the four exosomal muscle-specific miRNAs are associated with the progression of muscle wasting in DM1 patients. We propose that exosomal muscle-specific miRNAs may be useful molecular biomarkers for monitoring the progress of muscle wasting in DM1 patients. There has been a growing interest regarding the clinical applications of exosomes and their role in prognosis and therapy of various diseases and the above results contribute towards this way., (© The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

MicroRNAs (miRNAs) have been implicated in the development of some if not all cancer types and have been identified as attractive targets for prognosis, diagnosis, and therapy of the disease. miRNAs are a class of small non-coding RNAs (20-22 nt in length) that bind imperfectly to the 3'-untranslated region of target mRNA regulating gene expression. Aberrantly expressed miRNAs in cancer, sometimes known as oncomiRNAs, have been shown to play a major role in oncogenesis, metastasis, and drug resistance. Amplification of oncomiRNAs during cancer development correlates with the silencing of tumor suppressor genes; on the other hand, down-regulation of miRNAs has also been observed in cancer and cancer stem cells (CSCs). In both cases, miRNA regulation is inversely correlated with cancer progression. Growing evidence indicates that miRNAs are also involved in the metastatic process by either suppressing or promoting metastasis-related genes leading to the reduction or activation of cancer cell migration and invasion processes. In particular, circulating miRNAs (vesicle-encapsulated or non-encapsulated) have significant effects on tumorigenesis: membrane-particles, apoptotic bodies, and exosomes have been described as providers of a cell-to-cell communication system transporting oncogenic miRNAs from tumors to neighboring cells and distant metastatic sites. It is hypothesized that miRNAs control cancer development in a traditional manner, by regulating signaling pathways and factors. In addition, recent developments indicate a non-conventional mechanism of cancer regulation by stem cell reprograming via a regulatory network consisting of miRNAs and Wnt/β-catenin, Notch, and Hedgehog signaling pathways, all of which are involved in controlling stem cell functions of CSCs. In this review, we focus on the role of miRNAs in the Notch-pathway and how they regulate CSC self-renewal, differentiation and tumorigenesis by direct/indirect targeting of the Notch-pathway.

Rationale: MicroRNA (miRNA) biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far., Objective: To analyze changes in plasma miRNAs in response to antiplatelet therapy., Methods and Results: Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. In contrast, antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33), who received low-dose aspirin at baseline. Plasma levels of platelet miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition., Conclusions: Our study demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNAs responsive to antiplatelet therapy. It also highlights that antiplatelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma miRNAs to cardiovascular disease.

Objectives: This study sought to explore the association between baseline levels of microRNAs (miRNAs) (1995) and incident myocardial infarction (1995 to 2005) in the Bruneck cohort and determine their cellular origin., Background: Circulating miRNAs are emerging as potential biomarkers. We previously identified an miRNA signature for type 2 diabetes in the general population., Methods: A total of 19 candidate miRNAs were quantified by real-time polymerase chain reactions in 820 participants., Results: In multivariable Cox regression analysis, 3 miRNAs were consistently and significantly related to incident myocardial infarction: miR-126 showed a positive association (multivariable hazard ratio: 2.69 [95% confidence interval: 1.45 to 5.01], p = 0.002), whereas miR-223 and miR-197 were inversely associated with disease risk (multivariable hazard ratio: 0.47 [95% confidence interval: 0.29 to 0.75], p = 0.002, and 0.56 [95% confidence interval: 0.32 to 0.96], p = 0.036). To determine their cellular origin, healthy volunteers underwent limb ischemia-reperfusion generated by thigh cuff inflation, and plasma miRNA changes were analyzed at baseline, 10 min, 1 h, 5 h, 2 days, and 7 days. Computational analysis using the temporal clustering by affinity propagation algorithm identified 6 distinct miRNA clusters. One cluster included all miRNAs associated with the risk of future myocardial infarction. It was characterized by early (1 h) and sustained activation (7 days) post-ischemia-reperfusion injury and consisted of miRNAs predominantly expressed in platelets., Conclusions: In subjects with subsequent myocardial infarction, differential co-expression patterns of circulating miRNAs occur around endothelium-enriched miR-126, with platelets being a major contributor to this miRNA signature., (Copyright © 2012 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.)

The genetic epidemiology of Trichomonas vaginalis is poorly understood at present. The recent release of the organism's genome sequence opens the way to investigation of polymorphic markers allowing strain identification. We here report a preliminary analysis of microsatellite loci in T. vaginalis and show that this approach holds promise for future studies of infection transmission and organism diversity., (Copyright © 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.)

The concept of using stem cells for cardiovascular repair holds great potential, but uncertainties in preclinical experiments must be addressed before their therapeutic application. Contemporary proteomic techniques can help to characterize cell preparations more thoroughly and identify some of the potential causes that may lead to a high failure rate in clinical trials. The first part of this review discusses the broader application of proteomics to stem cell research by providing an overview of the main proteomic technologies and how they might help the translation of stem cell therapy. The second part focuses on the controversy about endothelial progenitor cells (EPCs) and raises cautionary flags for marker assignment and assessment of cell purity. A proteomics-led approach in early outgrowth EPCs has already raised the awareness that markers used to define their endothelial potential may arise from an uptake of platelet proteins. A platelet microparticle-related transfer of endothelial characteristics to mononuclear cells can result in a misinterpretation of the assay. The necessity to perform counterstaining for platelet markers in this setting is not fully appreciated. Similarly, the presence of platelets and platelet microparticles is not taken into consideration when functional improvements are directly attributed to EPCs, whereas saline solutions or plain medium serve as controls. Thus, proteomics shed new light on the caveats of a common stem cell assay in cardiovascular research, which might explain some of the inconsistencies in the field.

Early pro-angiogenic cells (EPCs) have been shown to be involved in neovascularization, angiogenesis and re-endothelialization and cathepsin L inhibition blunted their pro-angiogenic effect. In the present study, we have analysed and mapped the proteome and secretome of human EPCs, utilizing a combination of difference in-gel electrophoresis (DIGE) and shotgun proteomics. A population of 206 protein spots were analysed, with 171 being identified in the cellular proteome of EPCs. 82 proteins were identified in their conditioned medium, including the alternative macrophage markers C-C motif chemokine 18 (CCL18) and the hemoglobin scavenger receptor CD163 as well as platelet factor 4 (CXCL4) and platelet basic protein (CXCL7) with "platelet alpha granule" being returned as the top category according to the Gene Ontology Annotation. Apart from cathepsin L, the cathepsin L inhibitor also attenuated the release of a wide range of other cathepsins and lysosomal proteins such as legumain, but stimulated the secretion of members of the S100 protein family. The data presented here are the most comprehensive characterization of protein expression and secretion in human EPCs to date and highlight the potential importance of cysteine proteases in the processing of platelet factors for their pro-angiogenic potential. This article is part of a special issue entitled, "Cardiovascular Stem Cells Revisited"., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Rationale: MicroRNAs (miRNAs) have been implicated in the epigenetic regulation of key metabolic, inflammatory, and antiangiogenic pathways in type 2 diabetes (DM) and may contribute to common disease complications., Objective: In this study, we explore plasma miRNA profiles in patients with DM., Methods and Results: Total RNA was extracted from plasma samples of the prospective population-based Bruneck study. A total of 13 candidate miRNAs identified by microarray screening and miRNA network inference were quantified by quantitative PCR in all diabetic patients of the Bruneck study and age- and sex-matched controls (1995 evaluation, n=80 each). Quantitative PCR assessment revealed lower plasma levels of miR-20b, miR-21, miR-24, miR-15a, miR-126, miR-191, miR-197, miR-223, miR-320, and miR-486 in prevalent DM, but a modest increase of miR-28-3p. Findings emerged as robust in multivariable analysis and were independent of the standardization procedure applied. For endothelial miR-126, results were confirmed in the entire Bruneck cohort (n=822) in univariate (odds ratio [95% confidence interval], 0.38 [0.26 to 0.55]; P=2.72 × 10(-7)) and multivariate analyses (0.57 [0.37 to 0.86]; P=0.0082). Importantly, reduced miR-15a, miR-29b, miR-126, miR-223, and elevated miR-28-3p levels antedated the manifestation of disease. Most differences in miRNA levels were replicated in plasma obtained from hyperglycemic Lep(ob) mice. High glucose concentrations reduced the miR-126 content of endothelial apoptotic bodies. Similarly in patients with DM, the reduction of miR-126 was confined to circulating vesicles in plasma., Conclusions: We reveal a plasma miRNA signature for DM that includes loss of endothelial miR-126. These findings might explain the impaired peripheral angiogenic signaling in patients with DM.

Objective: Recent studies on cardiovascular progenitors have led to a new appreciation that paracrine factors may support the regeneration of damaged tissues., Methods and Results: We used a shotgun proteomics strategy to compare the secretome of peripheral blood-derived smooth muscle progenitors (SPCs) with human aortic smooth muscle cells. The late-outgrowth SPCs produced fewer proteolytic enzymes and inflammatory cytokines and showed reduced invasive capacity. Similar to smooth muscle cells, SPCs secreted extracellular matrix. However, SPCs produced different matrix proteins, as evidenced by the truncation of proangiogenic domains in collagen alpha-1 (I) and increased production of periostin. Moreover, SPCs retained serum proteins, including proteoglycans, regulating collagen assembly; and pigment epithelium-derived factor, a potent inhibitor of angiogenesis. As a functional consequence, their conditioned medium was less angiogenic, as demonstrated by endothelial tube formation assays in vitro and implantation of Matrigel plugs into nude, severe combined immunodeficient mice (NOD/SCID)., Conclusions: The present study represents an important conceptual development, suggesting that SPCs may contribute to extracellular matrix production.

The concept of endothelial progenitor cells (EPCs) has attracted considerable interest in cardiovascular research, but despite a decade of research there are still no specific markers for EPCs and results from clinical trials remain controversial. Using liquid chromatography-tandem mass spectrometry, we analyzed the protein composition of microparticles (MPs) originating from the cell surface of EPC cultures. Our data revealed that the conventional methods for isolating mononuclear cells lead to a contamination with platelet proteins. Notably, platelets readily disintegrate into platelet MPs. These platelet MPs are taken up by the mononuclear cell population, which acquires "endothelial" characteristics (CD31, von Willebrand factor [VWF], lectin-binding), and angiogenic properties. In a large population-based study (n = 526), platelets emerged as a positive predictor for the number of colony-forming units and early outgrowth EPCs. Our study provides the first evidence that the cell type consistent with current definitions of an EPC phenotype may arise from an uptake of platelet MPs by mononuclear cells resulting in a gross misinterpretation of their cellular progeny. These findings demonstrate the advantage of using an unbiased proteomic approach to assess cellular phenotypes and advise caution in attributing the benefits in clinical trials using unselected bone marrow mononuclear cells (BMCs) to stem cell-mediated repair.

Endothelial progenitor cell (EPC) cultures and colony-forming units (CFUs) have been extensively studied for their therapeutic and diagnostic potential. Recent data suggest a role for EPCs in the release of proangiogenic factors. To identify factors secreted by EPCs, conditioned medium from EPC cultures and CFUs was analyzed using a matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometer combined with offline peptide separation by nanoflow liquid chromatography. Results were verified by RT-PCR and multiplex cytokine assays and complemented by a cellular proteomic analysis of cultured EPCs and CFUs using difference in-gel electrophoresis. This extensive proteomic analysis revealed the presence of the proangiogenic factor thymidine phosphorylase (TP). Functional experiments demonstrated that inhibition of TP by 5-bromo-6-amino-uracil or gene silencing resulted in a significant increase in basal and oxidative stress-induced apoptosis, whereas supplementation with 2-deoxy-D-ribose-1-phosphate (dRP), the enzymatic product of TP, abrogated this effect. Moreover, dRP produced in EPC cultures stimulated endothelial cell migration in a paracrine manner, as demonstrated by gene-silencing experiments in transmigration and wound repair assays. RGD peptides and inhibitory antibodies to integrin alphavbeta3 attenuated the effect of conditioned medium from EPC cultures on endothelial migration. Finally, the effect of TP on angiogenesis was investigated by implantation of Matrigel plugs in mice. In these in vivo experiments, dRP strongly promoted neovascularization. Our data support the concept that EPCs exert their proangiogenic activity in a paracrine manner and demonstrate a key role of TP activity in their survival and proangiogenic potential.

The release of proteins and membrane vesicles in the bloodstream regulates diverse vascular processes, both physiological, such as angiogenesis and haemostasis, and pathological, such as atherosclerosis and atherothrombosis. Proteomics, beside its canonical application for the expression profiling in cells and organs, can be applied to the study of secreted proteins and microvesicles, which play a significant role in the homeostasis of the vasculature, and the development of the atherosclerotic disease., (Copyright © 2008 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)