Regulation of muscle satellite cell proliferation and differentiation by local trophic factors.

2012/2013The skeletal muscle is a terminally differentiated tissue. Its capacity to repair following injury or disease depends on a population of myogenic precursors, named satellite cells. These cells are localized beneath the skeletal muscle fiber, in a specialized microenvironment, the niche. The niche preserves the homeostatic conditions of satellite cell quiescence, but at the same time, it ensures their responsiveness to mechanical, physical and chemical triggers from the surrounding environment. Therefore, the composition of the external milieu is critical in determining satellite cell behavior. As a matter of fact, during aging or under pathological conditions, alterations of the extracellular environment entail a severe impairment of satellite cell ability to sustain regeneration and repair of the skeletal muscle tissue. The general goal of this thesis was to focus on some of the trophic factors potentially present in the satellite cell niche in vivo and to characterize their role on the modulation of satellite cell functions in vitro. The first part of the research activity dealt with the study of the trophic effect of ATP on mouse myoblast proliferation. From literature, it emerged that ATP is a potential regulator of the skeletal muscle regenerative program, however the signalling mechanism remained partially unknown. We observed that ATP increased myoblast growth rate, effect that was mimicked by low concentrations of H2O2. Reactive oxygen species (ROS) imaging revealed that ATP induced H2O2 production, at concentrations comparable to those effective in triggering myoblast proliferation. Interestingly, the exposure to equimolar concentrations of adenosine did not mimic the effect of ATP, excluding any role for the main hydrolysis product of ATP in the control of cell cycling. This result was in agreement with data reporting that the specific enzymes responsible for ATP degradation are poorly expressed in myoblasts and become upregulated after cell differentiation. In line with the latter observation, it appeared reasonable that the differentiating skeletal muscle cells were more exposed to ATP-derived adenosine than proliferating myoblasts, and this suggested a potential physiological role for the nucleoside adenosine in the later phases of myogenesis. Taking into account that adenosine receptors (ARs) are present in mouse myotubes, in a second study we hypothesized a crosstalk between nAChRs and ARs. Using the Ca2+-imaging technique, we observed that the pharmacological modulation of ARs triggered variations in the nAChR-driven ([Ca2+]i) spikes. Moreover, our preliminary results suggest not only an interplay between the two receptors but also that endogenous adenosine is tonically released by twitching myotubes and activates its receptors. The third research project was aimed at exploring the role of neural agrin, a heparan sulphate proteoglycan, so far known as the key organizer of post-synaptic elements during skeletal muscle differentiation/regeneration. Besides agrin’s canonical effect on the maturation of the NMJ, novel roles have been discovered in the recent years, suggesting that the neurotrophic factor has pleiotropic effects. In this new context, we pursued the identification of potential new roles for neural agrin in the determination of satellite cell behaviour. Firstly, the analysis of different cell models, including C2C12 cell line and primary mouse and human cells, and revealed an increase in IL-6 secretion following exposure to agrin. Secondly, we addressed the hypothesis of agrin as a potential modulator of human myoblasts proliferation. Our preliminary results demonstrate that agrin enhances the proliferative capacity of human satellite cells and suggest the potential mechanism involved in the signaling cascade.Il muscolo scheletrico è un tessuto terminalmente differenziato. La sua capacità rigenerativa in seguito a danno o patologia dipende da una popolazione di precursori miogenici, le cellule satelliti. Esse sono localizzate sulla superficie della fibra muscolare, racchiuse in un ambiente altamente specializzato, la nicchia. La nicchia assicura il mantenimento della quiescenza cellulare, ma allo stesso tempo fa sì che la cellula satellite risponda a stimoli meccanici, fisici o chimici, provenienti dall’ambiente esterno. Per questo motivo, la composizione dell’ambiente circostante condiziona altamente il comportamento della cellula satellite. Infatti, le alterazioni che si verificano con l’invecchiamento o in seguito a patologia compromettono la capacità dei precursori miogenici di sostenere la rigenerazione del tessuto. Lo scopo di questo lavoro di tesi è stato quello di individuare e caratterizzare alcuni dei fattori trofici che compongono il microambiente della cellula satellite in vivo, per cercare di capire come essi modulino le funzioni dei precursori miogenici durante la rigenerazione in vitro. La prima parte dell’attività di ricerca ha riguardato lo studio dell’effetto trofico dell’ATP sulla proliferazione mioblastica. Studi in letteratura hanno fatto emergere il potenziale ruolo regolatore dell’ATP nella rigenerazione muscolare, anche se i meccanismi attraverso cui opera non sono ancora chiari. I risultati da noi ottenuti hanno dimostrato che l’ATP aumenta la proliferazione mioblastica e che un effetto simile si osserva in presenza di H2O2. L’imaging per le specie reattive dell’ossigeno (ROS) ha inoltre dimostrato che l’ATP induce la produzione di H2O2, a concentrazioni paragonabili a quelle capaci di aumentare la proliferazione. In presenza di concentrazioni equimolari di adenosina non è stato osservato alcun effetto sulla proliferazione cellulare, fatto che suggerisce che il ruolo dell’ATP non sia attribuibile all’adenosina, il suo principale prodotto di degradazione. Questo risultato è in accordo con quanto già riportato da altri Autori a proposito degli enzimi responsabili dell’idrolisi dell’ATP: essi sono poco espressi nei mioblasti in proliferazione, mentre la loro espressione aumenta notevolmente con il differenziamento cellulare. Alla luce di queste osservazioni, risultava verosimile che i miotubi fossero più esposti rispetto ai mioblasti all’adenosina derivante dall’ATP e che pertanto l’adenosina avesse un ruolo fisiologico preponderante nelle fasi avanzate della miogenesi. Dal momento che i recettori per l’adenosina (ARs) sono espressi nei miotubi murini, un secondo lavoro ha avuto come obiettivo quello di investigare un possibile “crosstalk” tra i ARs e i nAChRs. Esperimenti di Ca2+- imaging hanno dimostrato come la modulazione farmacologica dei ARs si traduca in una variazione nelle oscillazioni di [Ca2+]i indotte dall’attività del nAChR. Questi risultati, sebbene preliminari, suggeriscono non solo che i due recettori interagiscono tra loro, ma anche che l’adenosina è tonicamente secreta dai miotubi in contrazione e agisca attivando i suoi recettori. Il terzo progetto di ricerca è stato finalizzato allo studio del ruolo dell’agrina neuronale, un proteoglicano eparan solfato, già noto per la sua capacità di aggregare elementi sinaptici durante la fase di differenziamento e di rigenerazione del muscolo scheletrico. Accanto al ruolo canonico che la vede coinvolta nella maturazione della giunzione neuromuscolare, negli ultimi anni sono state descritte nuove funzioni per l’agrina neuronale, che la indicano come un fattore pleiotropico. In questo contesto, abbiamo esplorato nuove proprietà dell’agrina. In primo luogo, l’analisi di diversi modelli cellulari, incluse la linea cellulare C2C12 e cellule primarie murine e umane, ha dimostrato che il fattore neurotrofico potenzia il rilascio di IL-6. In un secondo studio, è stato ipotizzato un potenziale effetto di modulazione della proliferazione di mioblasti umani da parte dell’agrina neuronale. Risultati preliminari hanno dimostrato che l’agrina aumenta la capacità proliferativa delle cellule satelliti umane. Inoltre, sono stati individuati alcuni dei fattori molecolari che partecipano alla cascata di segnalazione.XXVI Ciclo198

Plasma Technology Reduces Blood Loss in Adolescent Idiopathic Scoliosis Surgery: A Prospective Randomized Clinical Trial

Study Design: Prospective randomized clinical trial. Objectives: To assess the effectiveness of PEAK Plasmablade (PPB), compared with bipolar sealer and standard electrocautery, in the posterior spinal instrumentation and fusion (PSF) surgery performed for adolescent idiopathic scoliosis (AIS). Methods: Ninety-three patients undergoing PSF surgery for AIS were randomized in 2 groups: group-A patients (n = 45) underwent PSF surgery using PPB; group-B patients (n = 48) were treated with bipolar sealer and standard electrocautery. Demographic and surgical data was recorded. All the patients underwent serial blood tests on the day before surgery (T0) and at 24 (T1), 48 (T2), 72 (T3), and 96 (T4) hours postoperatively. Visual analogue scale for pain (VAS) score, the percentage of paracetamol assumption, and the blood transfusion rate were recorded in the time-lapse T1 to T4. Intergroup variability was assessed. Pearson correlation test was performed. A P value <.05 was considered significant. Results: In group A, a significantly shorter total operative time ( P = .0087), a significantly lower total intraoperative blood loss (TBL) ( P = .001), and a higher postoperative hemoglobin (Hb) ( P = .01) were recorded. A significant higher mean Hb concentration and mean albumin value was recorded in group A at 24 and 48 hours postoperatively. A significant correlation between TBL and hospital stay was recorded in both groups (group A, P = .00 001; group B, P = .00 006); moreover, in both groups, a significant correlation was observed between TBL and mean VAS at 72 hours postoperatively (group A, P = .0009; group B, P = .0001) and at 96 hours postoperatively (group A, P = .000 044; group B, P = .00 001). Conclusions: PPB reduces the intraoperative blood loss in PSF performed for AIS, thus allowing a patient's faster recovery

Multi-valve Libman-Sacks’s endocarditis-related multiple, massive and fatal systemic embolization. A case report and a review of diagnostic work-up

We reported a case of non-bacterial thrombotic endocarditis (NBTE) in a 37-year-old woman who presented with signs and symptoms of cardio-embolic cerebral stroke caused by a prothrombotic state due to underlying advanced uterine cancer. Multimodal imaging, including 3D-ecocardiography, as well as laboratory and cultural tests, were critical in making the diagnosis. After starting anticoagulation therapy with low molecular weight heparin (LMWH), the patient underwent surgical aortic valve replacement due to worsening aortic valve function, initial left ventricle enlargement, increasing dimensions, and mobility of vegetations. Unfortunately, vegetations relapsed on the aortic valve bio-prosthesis as well as the mitral leaflets, resulting in a final picture of multi-valve NBTE. The fatal outcome was due to a massive multiple limb embolism, which resulted in leg amputations and septical complications. Starting with the case, we present a brief overview of the pathology's presentation, treatment, management, and prognosis, as well as the diagnostic work-up

Neuronal Agrin Promotes Proliferation of Primary Human Myoblasts in an Age-Dependent Manner

Neuronal agrin, a heparan sulphate proteoglycan secreted by the -motor neurons, promotes the formation and maintenance of the neuromuscular junction by binding to Lrp4 and activating muscle-specific kinase (MuSK). Neuronal agrin also promotes myogenesis by enhancing differentiation and maturation of myotubes, but its effect on proliferating human myoblasts, which are often considered to be unresponsive to agrin, remains unclear. Using primary human myoblasts, we determined that neuronal agrin induced transient dephosphorylation of ERK1/2, while c-Abl, STAT3, and focal adhesion kinase were unresponsive. Gene silencing of Lrp4 and MuSK markedly reduced the BrdU incorporation, suggesting the functional importance of the Lrp4/MuSK complex for myoblast proliferation. Acute and chronic treatments with neuronal agrin increased the proliferation of human myoblasts in old donors, but they did not affect the proliferation of myoblasts in young donors. The C-terminal fragment of agrin which lacks the Lrp4-binding site and cannot activate MuSK had a similar age-dependent effect, indicating that the age-dependent signalling pathways activated by neuronal agrin involve the Lrp4/MuSK receptor complex as well as an Lrp4/MuSK-independent pathway which remained unknown. Collectively, our results highlight an age-dependent role for neuronal agrin in promoting the proliferation of human myoblasts

Cooper-Frye Formula and Non-extensive Coalescence at RHIC Energy

Transverse spectra are calculated for various types of hadrons stemming from Au Au collisions at $\sqrt{s}=200$ GeV. We utilize a quark recombination model based on generalized Boltzmann-Gibbs thermodynamics for local hadron production at various break-up scenarios.Comment: 4 pages, 1 figur

Unlocking insights in bicuspid aortic valve management in adult patients: the vital role of cardiac imaging

The bicuspid aortic valve (BAV) presents a multifaceted clinical challenge due to its diverse morphologies and associated complications. This review aims to elucidate the critical role of cardiac imaging in guiding optimal management strategies for BAV patients. BAV, with a prevalence of 1% to 2%, has genetic underpinnings linked to the NOTCH1 gene mutation. Variability in BAV morphology necessitates tailored surgical approaches. The three primary types of BAV morphology - right-left cusp fusion, right-noncoronary cusp fusion, and left-noncoronary cusp fusion - demand nuanced considerations due to their distinct implications. Valvular dysfunction results in aortic stenosis or regurgitation, attributed to altered valve structure and turbulent hemodynamics. Cardiac imaging modalities, including echocardiography, magnetic resonance imaging, and computerized tomography, are instrumental in assessing valve function, aortic dimensions, and associated complications. Imaging helps predict potential complications, enabling informed treatment decisions. Regular follow-up is crucial to detecting alterations early and intervening promptly. Surgical management options encompass aortic valve repair or replacement, with patient-specific factors guiding the choice. Post-surgical surveillance plays a vital role in preventing complications and optimizing patient outcomes. The review underscores the significance of advanced cardiac imaging techniques in understanding BAV's complexities, facilitating personalized management strategies, and improving patient care. By harnessing the power of multimodal imaging, clinicians can tailor interventions, monitor disease progression, and ultimately enhance the prognosis and quality of life for individuals with BAV

Tubulin tyrosination regulates synaptic function and is disrupted in Alzheimer's disease

: Microtubules play fundamental roles in the maintenance of neuronal processes and in synaptic function and plasticity. While dynamic microtubules are mainly composed of tyrosinated tubulin, long-lived microtubules contain detyrosinated tubulin, suggesting that the tubulin tyrosination/detyrosination cycle is a key player in the maintenance of microtubule dynamics and neuronal homeostasis, conditions which go awry in neurodegenerative diseases. In the tyrosination/detyrosination cycle, the C-terminal tyrosine of α-tubulin is removed by tubulin carboxypeptidases and re-added by tubulin tyrosine ligase. Here we show that tubulin tyrosine ligase hemizygous mice exhibit decreased tyrosinated microtubules, reduced dendritic spine density, and both synaptic plasticity and memory deficits. We further report decreased tubulin tyrosine ligase expression in sporadic and familial Alzheimer's disease, and reduced microtubule dynamics in human neurons harboring the familial APP-V717I mutation. Finally, we show that synapses visited by dynamic microtubules are more resistant to oligomeric amyloid β peptide toxicity and that expression of tubulin tyrosine ligase, by restoring microtubule entry into spines, suppresses the loss of synapses induced by amyloid β peptide. Together, our results demonstrate that a balanced tyrosination/detyrosination tubulin cycle is necessary for the maintenance of synaptic plasticity, is protective against amyloid β peptide-induced synaptic damage, and that this balance is lost in Alzheimer's disease, providing evidence that defective tubulin retyrosination may contribute to circuit dysfunction during neurodegeneration in Alzheimer's disease

Effects of Newcastle Disease Virus Strains AF2240 and V4-UPM on Cytolysis and Apoptosis of Leukemia Cell Lines

Newcastle disease virus (NDV) is used as an antineoplastic agent in clinical tumor therapy. It has prompted much interest as an anticancer agent because it can replicate up to 10,000 times better in human cancer cells than in most normal cells. This study was carried out to determine the oncolytic potential of NDV strain AF2240 and V4-UPM on WEHI-3B leukemia cell line. Results from MTT cytotoxicity assay showed that the CD50 values for both strains were 2 and 8 HAU for AF2240 and V4-UPM, respectively. In addition, bromodeoxyuridine (BrdU) and trypan blue dye exclusion assays showed inhibition in cell proliferation after different periods. Increase in the cellular level of caspase-3 and detection of DNA laddering using agarose gel electrophoresis on treated cells with NDV confirmed that the mode of cell death was apoptosis. In addition, flow-cytometry analysis of cellular DNA content showed that the virus caused an increase in the sub-G1 region (apoptosis peaks). In conclusion, NDV strains AF2240 and V4-UPM caused cytolytic effects against WEHI-3B leukemic cell line