In the past few years, skeletal muscle has emerged as an important secretory organ producing soluble factors, called myokines, that exert either autocrine, paracrine or endocrine effects. Moreover, ...recent studies have shown that muscle releases microRNAs into the bloodstream in response to physical exercise. These microRNAs affect target cells, such as hormones and cytokines. The mechanisms underlying microRNA secretion are poorly characterized at present. Here, we investigated whether muscle tissue releases extracellular vesicles (EVs), which carry microRNAs in the bloodstream under physiological conditions such as physical exercise. Using density gradient separation of plasma from sedentary and physically fit young men we found EVs positive for TSG101 and alpha-sarcoglycan (SGCA), and enriched for miR-206. Cytometric analysis showed that the SGCA+ EVs account for 1-5% of the total and that 60-65% of these EVs were also positive for the exosomal marker CD81. Furthermore, the SGCA-immuno captured sub-population of EVs exhibited higher levels of the miR-206/miR16 ratio compared to total plasma EVs. Finally, a significant positive correlation was found between the aerobic fitness and muscle-specific miRNAs and EV miR-133b and -181a-5p were significantly up-regulated after acute exercise. Thus, our study proposes EVs as a novel means of muscle communication potentially involved in muscle remodeling and homeostasis.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Neuromuscular junction (NMJ) formation involves morphological changes both in motor terminals and muscle membrane. The molecular mechanisms leading to NMJ formation and maintenance have not yet been ...fully elucidated. During the last decade, it has become clear that virtually all cells release different types of extracellular vesicles (EVs), which can be taken up by nearby or distant cells modulating their activity. Initially, EVs were associated to a mechanism involved in the elimination of unwanted material; subsequent evidence demonstrated that exosomes, and more in general EVs, play a key role in intercellular communication by transferring proteins, lipids, DNA and RNA to target cells. Recently, EVs have emerged as potent carriers for Wnt, bone morphogenetic protein, miRNA secretion and extracellular traveling. Convincing evidence demonstrates that presynaptic terminals release exosomes that are taken up by muscle cells, and these exosomes can modulate synaptic plasticity in the recipient muscle cell in vivo. Furthermore, recent data highlighted that EVs could also be a potential cause of neurodegenerative disorders. Indeed, mutant SOD1, TDP-43 and FUS/TLS can be secreted by neural cells packaged into EVs and enter in neighboring neural cells, contributing to the onset and severity of the disease.
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Growing evidence points to the effectiveness of flywheel (FW) based iso-inertial resistance training in improving physical performance capacities. However, molecular adaptations induced by FW ...exercises are largely unknown. Eight resistance-trained men performed 5 sets of 10 maximal squats on a FW device. Muscle biopsies (fine needle aspiration technique) and blood samples were collected before (t0), and 2 h (t1) after FW exercise. Blood samples were additionally drawn after 24 h (t2) and 48 h (t3). Paired samples
-tests revealed significant increases, at t1, of mRNA expression of the genes involved in inflammation, in both muscle (
α
) and peripheral blood mononuclear cells (
α
). Circulating extracellular vesicles (EVs) and EV-encapsulated miRNA levels (miR-206, miR-146a) significantly increased at t1 as well. Conversely, muscle mRNA level of genes associated with muscle growth/remodeling (
) decreased at t1. One-way repeated measure ANOVAs, with Bonferroni corrected
pairwise comparisons, revealed significant increases in plasma concentrations of IL-6 (t1; t2; t3) and muscle creatine kinase (t1; t2), while IGF-1 significantly increased at t2 only. Our findings show that, even in experienced resistance trained individuals, a single FW training session modifies local and systemic markers involved in late structural remodeling and functional adaptation of skeletal muscle.
The role of epigenetics in endothelial cell senescence is a cutting-edge topic in ageing research. However, little is known of the relative contribution to pro-senescence signal propagation provided ...by microRNAs shuttled by extracellular vesicles (EVs) released from senescent cells. Analysis of microRNA and DNA methylation profiles in non-senescent (control) and senescent (SEN) human umbilical vein endothelial cells (HUVECs), and microRNA profiling of their cognate small EVs (sEVs) and large EVs demonstrated that SEN cells released a significantly greater sEV number than control cells. sEVs were enriched in miR-21-5p and miR-217, which target DNMT1 and SIRT1. Treatment of control cells with SEN sEVs induced a miR-21/miR-217-related impairment of DNMT1-SIRT1 expression, the reduction of proliferation markers, the acquisition of a senescent phenotype and a partial demethylation of the locus encoding for miR-21. MicroRNA profiling of sEVs from plasma of healthy subjects aged 40-100 years showed an inverse U-shaped age-related trend for miR-21-5p, consistent with senescence-associated biomarker profiles. Our findings suggest that miR-21-5p/miR-217 carried by SEN sEVs spread pro-senescence signals, affecting DNA methylation and cell replication.
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FZAB, GIS, IJS, KILJ, NLZOH, NUK, OILJ, SAZU, SBCE, SBMB, UL, UM, UPUK
Myogenic differentiation is triggered, among other situations, in response to muscle damage for regenerative purposes. It has been shown that during myogenic differentiation, myotubes release ...extracellular vesicles (EVs) which participate in the signalling pattern of the microenvironment. Here we investigated whether EVs released by myotubes exposed or not to mild oxidative stress modulate the behaviour of targeted differentiating myoblasts and macrophages to promote myogenesis. We found that EVs released by oxidatively challenged myotubes (H₂O₂-EVs) are characterized by an increased loading of nucleic acids, mainly DNA. In addition, incubation of myoblasts with H₂O₂-EVs resulted in a significant decrease of myotube diameter, myogenin mRNA levels and myosin heavy chain expression along with an upregulation of proliferating cell nuclear antigen: these effects collectively lead to an increase of recipient myoblast proliferation. Notably, the EVs from untreated myotubes induced an opposite trend in myoblasts, that is, a slight pro-differentiation effect. Finally, H₂O₂-EVs were capable of eliciting an increased interleukin 6 mRNA expression in RAW264.7 macrophages. Notably, this is the first demonstration that myotubes communicate with surrounding macrophages via EV release. Collectively, the data reported herein suggest that myotubes, depending on their conditions, release EVs carrying differential signals which could contribute to finely and coherently orchestrate the muscle regeneration process.
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Objective The intensity of barbell bench press exercise is generally prescribed as the load to be lifted for a specific number of repetitions; however, other factors (e.g., execution velocity) can ...affect bench press exercise intensity. Moreover, no study assessed whether load distribution (i.e., the distance between the disc stacks on the two sides of the barbell) affects exercise intensity. The present study aims to assess how different combinations of load, velocity, and barbell load distribution affect the number of repetitions to failure (REPfailure), and rating of perceived exertion (RPEfatigue) and number of repetitions (REPfatigue) at fatigue onset. Methods Ten males (age 23.3±1.8 years) performed bench press exercises to exhaustion using random combinations of three loads (50%, 65%, and 80% of 1 repetition maximum), three execution velocities (50%, 70%, and 90% of maximal concentric velocity), and two load distributions (narrow and wide). Three separate three-way repeated-measures ANOVAs were performed to assess the effect of load, velocity, and load distribution on REPfailure, RPEfatigue, and REPfatigue expressed as a percentage of REPfailure. Results REPfailure was affected by load (p<0.001), velocity (p<0.001), and distribution (p = 0.005). The interactions between load and velocity (p<0.001) and load and distribution (p = 0.004) showed a significant effect on REPfailure, whereas the interaction between velocity and distribution was not significant (p = 0.360). Overall, more REPfailure were performed using lower loads, higher velocities, and a wider distribution. RPEfatigue and REPfatigue were affected by load (p<0.001 and p = 0.007, respectively) and velocity (p<0.001 and p<0.001, respectively), and not by distribution (p = 0.510 and p = 0.571, respectively) or the two-way interaction effects. Overall, using higher loads yielded higher RPEfatigue but lower REPfatigue, while RPEfatigue and REPfatigue were higher when slower velocities were used. Conclusion The current investigation shows that not only load but also velocity and barbell load distribution may influence bench press training volume and perceived exertion.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The intensity of barbell bench press exercise is generally prescribed as the load to be lifted for a specific number of repetitions; however, other factors (e.g., execution velocity) can affect bench ...press exercise intensity. Moreover, no study assessed whether load distribution (i.e., the distance between the disc stacks on the two sides of the barbell) affects exercise intensity. The present study aims to assess how different combinations of load, velocity, and barbell load distribution affect the number of repetitions to failure (REPfailure), and rating of perceived exertion (RPEfatigue) and number of repetitions (REPfatigue) at fatigue onset.
Ten males (age 23.3±1.8 years) performed bench press exercises to exhaustion using random combinations of three loads (50%, 65%, and 80% of 1 repetition maximum), three execution velocities (50%, 70%, and 90% of maximal concentric velocity), and two load distributions (narrow and wide). Three separate three-way repeated-measures ANOVAs were performed to assess the effect of load, velocity, and load distribution on REPfailure, RPEfatigue, and REPfatigue expressed as a percentage of REPfailure.
REPfailure was affected by load (p<0.001), velocity (p<0.001), and distribution (p = 0.005). The interactions between load and velocity (p<0.001) and load and distribution (p = 0.004) showed a significant effect on REPfailure, whereas the interaction between velocity and distribution was not significant (p = 0.360). Overall, more REPfailure were performed using lower loads, higher velocities, and a wider distribution. RPEfatigue and REPfatigue were affected by load (p<0.001 and p = 0.007, respectively) and velocity (p<0.001 and p<0.001, respectively), and not by distribution (p = 0.510 and p = 0.571, respectively) or the two-way interaction effects. Overall, using higher loads yielded higher RPEfatigue but lower REPfatigue, while RPEfatigue and REPfatigue were higher when slower velocities were used.
The current investigation shows that not only load but also velocity and barbell load distribution may influence bench press training volume and perceived exertion.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Exercise-released extracellular vesicles (EVs) are emerging as a novel class of exerkines that promotes systemic beneficial effects. However, slight differences in the applied exercise protocols in ...terms of mode, intensity and duration, as well as the need for standardized protocols for EV isolation, make the comparison of the studies in the literature extremely difficult. This work aims to investigate the EV amount and EV-associated miRNAs released in circulation in response to different physical exercise regimens. Healthy individuals were subjected to different exercise protocols: acute aerobic exercise (AAE) and training (AT), acute maximal aerobic exercise (AMAE) and altitude aerobic training (AAT). We found a tendency for total EVs to increase in the sedentary condition compared to trained participants following AAE. Moreover, the cytofluorimetric analysis showed an increase in CD81
/SGCA
/CD45
EVs in response to AAE. Although a single bout of moderate/maximal exercise did not impact the total EV number, EV-miRNA levels were affected as a result. In detail, EV-associated miR-206, miR-133b and miR-146a were upregulated following AAE, and this trend appeared intensity-dependent. Finally, THP-1 macrophage treatment with exercise-derived EVs induced an increase of the mRNAs encoding for
,
and
using baseline and immediately post-exercise EVs. Still, 1 h post-exercise EVs failed to stimulate a pro-inflammatory program. In conclusion, the reported data provide a better understanding of the release of circulating EVs and their role as mediators of the inflammatory processes associated with exercise.
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Plant-derived nanovesicles (PDNVs) have become attractive alternatives to mammalian cell-derived extracellular vesicles (EVs) both as therapeutic approaches and drug-delivery vehicles. In this study, ...we isolated tomato fruit-derived NVs and separated them by the iodixanol density gradient ultracentrifugation (DGUC) into twelve fractions. Three visible bands were observed at densities 1.064 ± 0.007 g/mL, 1.103 ± 0.006 g/mL and 1.122 ± 0.012 g/mL. Crude tomato PDNVs and DGUC fractions were characterized by particle size-distribution, concentration, lipid and protein contents as well as protein composition using mass spectrometry-based proteomics. Cytotoxicity and anti-inflammatory activity of the DGUC fractions associated to these bands were assessed in the lipopolysaccharide (LPS)-stimulated human monocytic THP-1 cell culture. The middle and the low-density visible DGUC fractions of tomato PDNVs showed a significant reduction in LPS-induced inflammatory IL-1β cytokine mRNA production. Functional analysis of proteins identified in these fractions reveals the presence of 14-3-3 proteins, endoplasmic reticulum luminal binding proteins and GTP binding proteins associated to gene ontology (GO) term GO:0050794 and the regulation of several cellular processes including inflammation. The most abundant middle-density DGUC fraction was loaded with curcumin using direct loading, sonication and extrusion methods and anti-inflammatory activity was compared. The highest entrapment efficiency and drug loading capacity was obtained by direct loading. Curcumin loaded by sonication increased the basal anti-inflammatory activity of tomato PDNVs.
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Extracellular vesicles (EVs) are small lipid particles secreted by almost all human cells into the extracellular space. They perform the essential function of cell-to-cell communication, and their ...role in promoting breast cancer progression has been well demonstrated. It is known that EVs released by triple-negative and highly aggressive MDA-MB-231 breast cancer cells treated with paclitaxel, a microtubule-targeting agent (MTA), promoted chemoresistance in EV-recipient cells. Here, we studied the RNA content of EVs produced by the same MDA-MB-231 breast cancer cells treated with another MTA, eribulin mesylate. In particular, we analyzed the expression of different RNA species, including mRNAs, lncRNAs, miRNAs, snoRNAs, piRNAs and tRNA fragments by RNA-seq. Then, we performed differential expression analysis, weighted gene co-expression network analysis (WGCNA), functional enrichment analysis, and miRNA-target identification. Our findings demonstrate the possible involvement of EVs from eribulin-treated cells in the spread of chemoresistance, prompting the design of strategies that selectively target tumor EVs.
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