Angiogenic role of extracellular vesicles in human platelet lysate

Il principale ruolo delle piastrine è conosciuto in processi quali l’emostasi e la trombosi, ma esse svolgono la loro funzione anche come principali regolatori dell'angiogenesi, costituita da fasi di proliferazione endoteliale, germinazione e neo-angiogenesi1-3. L'angiogenesi è un processo chiave per la rigenerazione dei tessuti e il ruolo emergente delle piastrine nel migliorare l'angiogenesi deriva dalla loro interazione con l'endotelio, ad esempio durante il danno vascolare dove agiscono per preservare l'integrità e l'omeostasi dei vasi 2. Inoltre, le piastrine mostrano un profilo secretorio unico composto da molteplici fattori di crescita, citochine, microRNA, piccole molecole solubili e proteine con un doppio ruolo pro e antiangiogenico 3-5. Questa combinazione equilibrata di mediatori è principalmente contenuta in vescicole extracellulari (EV), oggi concepite come segnalosomi o vettori biologici delle quali le piastrine rappresentano la fonte più abbondante dell’organismo umano 6,7. Le EV possono essere di dimensioni e composizione eterogenee (esosomi, microparticelle, macrovesicle) ed è stato riportato che rispecchiano le proprietà emostatiche delle piastrine8. Il coinvolgimento delle piastrine e delle EV nei processi angiogenici in cellule endoteliali ha incoraggiato una migliore comprensione del loro potenziale terapeutico per applicazioni di tipo rigenerativo in cui il ripristino o il miglioramento dell'angiogenesi rappresenta l’obiettivo clinico da raggiungere9. Di conseguenza anche il preparato clinico conosciuto come lisato piastrinico (LP) è in grado di potenziare e riflettere le proprietà angiogeniche delle piastrine4. La pletora di fattori, molecole solubili ed EV altamente concentrati in questo emoderivato è considerata la principale responsabile della sua capacità angiogenica, ma la modalità con cui l'EV derivate dal LP potrebbero regolare l'angiogenesi deve ancora essere affrontata in modo completo. A tal proposito, lo scopo del mio studio è confermare che LP Mesengen®, (brevetto No.WO2013042095) rappresenta una fonte di EV biodisponibile e quindi validare il ruolo individuale delle EV in relazione alla capacità di mediare l'angiogenesi in cellule endoteliali. Inoltre, vogliamo dimostrare quali meccanismi molecolari ed azioni biologiche mediate dal LP e dalle sole EV derivate dal LP possono contribuire al ripristino dell’ endotelio dopo insulto e quindi aprire una nuova strada sull’impiego del LP come strumento per la rigenerazione vascolare nell’ambito delle cell-free therapy. I risultati ottenuti mostrano che il LP contiene EV, ottenute mediante tecnica di ultracentrifugazione (100000g per 1h) e caratterizzate in base a dimensione, fenotipo e morfologia10. Infatti, grazie all’ausilio di filtri 0,8 e 0,22 m e beads a dimensione nota, l’analisi citofluorimetrica FACS ha dimostrato la presenza di classi di EV eterogenee con diametro di >800 nm, compreso tra 800 nm e 200 nm e < 200 nm. Le EV sono state caratterizzate ulteriormente valutando il loro fenotipo, risultato principalmente positivo per il CD41 (marcatore proteico della membrana piastrinica o glicoproteina IIb, 50,61% ) e un espressione molto bassa del doppio positivo CD41/CD61(canonico marcatore esosomiale, 0,76%) 11,12. L’osservazione morfologica tramite TEM ha rivelato la presenza delle medesime vescicole dimensionalmente eterogenee, a forma tondeggiante ed elettrondense, fattore che indica la presenza di un contenuto vescicolare11. I nostri risultati in vitro, su HUVEC (Human Umbilical Vein Endothelial Cell) trattate con EV derivate dal 10%LP per 24h, hanno dimostrato la loro interazione a livello citoplamatico. Inoltre, le diverse filtrazioni del LP, pur non influenzando migrazione e proliferazione, sono in grado di alterare l’attività angiogenica delle cellule endoteliali. In particolar modo abbiamo osservato una diminuzione significativa della capacità di formare loop nel saggio Matrigel (p<0.001) dopo la somministrazione del LP 0,22, contenente quindi EV inferiori a 200nm. Per confermare che le sole EV sono responsabili dell’effetto angiogenico sono state estratte e somministrate alle HUVEC durante il medesimo saggio Matrigel10. I risultati ottenuti hanno dimostrato che le EV 0,22 hanno ridotto significativamente l'effetto angiogenico rispetto al 10% EV (p=0,04), che è stato a sua volta in grado di aumentare il numero di loop rispetto alla condizione senza siero ( EBM p=0,015) , ma ancora in misura minore rispetto al 10% LP (p=0,0198). È interessante notare che, in condizioni di ipossia l’effetto angiogenico è stato conservato in tutte le condizioni. Inoltre, abbiamo corroborato questa osservazione tramite il 3D bioprinting con fibre di idrogel in grado di incorporare le HUVEC sottoposte al trattamento con i diversi terreni EGM-2 (controllo positivo), EBM (controllo negativo), 10%LP, LP 0,8 e LP 0,22. È stato quindi dimostrato che le strutture simili a vasi, risultanti dal 3D bioprinting, presentano una percentuale maggiore dell'area endoteliale CD31+/vWF + nei trattamento 10%LP rispetto a LP 0,22 (p=0,0382). Inoltre il mezzo condizionato con LP 0,8 e LP 0,22 diminuisce significativamente i livelli di perossido di idrogeno (H2O2) rispetto a 10% LP. Poiché Nox4 è considerato la principale e specifica isoforma NADPH responsabile della produzione diretta di H2O2 nelle cellule endoteliali13,14, abbiamo studiato il suo potenziale coinvolgimento, notando che LP 0,22 è in grado di migliorare i livelli di mRNA Nox4 rispetto al 10% LP (p=0,0021) . Il condizionamento ipossico è stato in grado di equalizzare i livelli espressione di Nox4 in tutte le condizioni (p>0,05 tutto ). A questo punto, abbiamo nuovamente trattato le colture direttamente con le EV ultracentrifugate e i risultati hanno mostrato che l'intero pool del 10%EV ha ridotto i livelli di mRNA di Nox4 rispetto al 10% LP (p=0,04), ma la rimozione della frazione di EV>200nm dall'intero pool di EV non ha modificato questo scenario, suggerendo che l'intero pool di vescicole probabilmente contribuisce alla modulazione di Nox4. È interessante notare che l' ipossia ha ribaltato questo scenario, appiattendo queste differenze tra le condizioni (p>0,05 tutte). Inoltre, data la presenza in letteratura di studi sulla funzionalità piastrinica mediata da microRNA e il loro trasporto correlato a funzioni vescicolari, abbiamo analizzato, per la prima volta, il miRNoma del LP tramite RNA Seq. Ne è risultato un contenuto di RNA, rappresentato da miRNA (43%), seguito da Y RNA (17%), RNA antisenso (10%) e lincRNA (8 %). Successive analisi in silico, ci hanno permesso di selezionare miRNA altamente espressi e coerenti tra i 4 replicati di LP analizzati e di correrali, grazie a database di ontologia genica, a miRNA noti coinvolti nell’ attivazione endoteliale, nella vasculogenesi e nell’ossidazione NADPH . Cosi da ottenere una lista di 5 miRNA: miR-320a; miR-148b-3p; miR-25-3p; miR-26b-5p; miR-152-3p. Inoltre, abbiamo scoperto che miR126 , noto anche come angiomiRNA ed espresso selettivamente sulle cellule endoteliali15, era tra i primi 40 miRNA espressi nel LP. Di conseguenza, abbiamo validato la sua presenza nel nostro LP riscontrando livelli di miR 126 tramite Real Time PCR che aumentano in modo proporzionale alla quantità di LP testato (p=0,043). In conclusione possiamo affermare che il LP Mesengen contiene EV di origine pistrinica con dimensioni differenti ed eterogenee che possiedono attività angiogeniche. Il nostro LP è in grado di modulare lo stato ossidativo di cellule endoteliali attraverso l’interazione con il pathway della Nox4. Importanti analisi di Small RNA seq hanno fatto emergere la presenza nel LP di miRNA, espressi in modo omogeneo, verosimilmente correlati a funzioni endoteliale, vascolari e stato ossidativo NADPH. Abbreviazioni LP= Lisato piastrinico; EV= vescicole extracellulari; LP 0,8= lisato piastrinico al 10% filtrato 0,8 m; LP 0,22= lisato piastrinico al 10% filtrato 0,22 m; EV 0,8= vescicole extracellulari al 10% filtrate 0,8 m; EV 0,22= vescicole extracellulari filtrate 0,22 m

Focal points for a more user-centred agile development

The integration of user-centred design and Agile development is becoming increasingly common in companies and appears promising. However it may also present some critical points, or communication breakdowns, such as a variable interpretation of user involvement, a mismatch in the value of documentation and a misalignment in iterations. We refine these themes, emerging from both literature and previous fieldwork, by analysing a case study performed in an IT company that adopts both software engineering approaches, and we further extend the framework with a new theme related to task ownership. We argue that communication breakdowns can become focal points to drive action and decision for establishing an organisational context acknowledging the value of user involvement: to this end, we suggest the adoption of design thinking and the active engagement of the customer in embracing its values

Getting old through the blood. Circulating molecules in aging and senescence of cardiovascular regenerative cells

Global aging is a hallmark of our century. The natural multifactorial process resulting in aging involves structural and functional changes, affecting molecules, cells, and tissues. As the western population is getting older, we are witnessing an increase in the burden of cardiovascular events, some of which are known to be directly linked to cellular senescence and dysfunction. In this review, we will focus on the description of a few circulating molecules, which have been correlated to life span, aging, and cardiovascular homeostasis. We will review the current literature concerning the circulating levels and related signaling pathways of selected proteins (insulin-like growth factor 1, growth and differentiation factor-11, and PAI-1) and microRNAs of interest (miR-34a, miR-146a, miR-21), whose bloodstream levels have been associated to aging in different organisms. In particular, we will also discuss their potential role in the biology and senescence of cardiovascular regenerative cell types, such as endothelial progenitor cells, mesenchymal stromal cells, and cardiac progenitor cells

Oral plaque from Type 2 diabetic patients reduces the clonogenic capacity of dental pulp-derived mesenchymal stem cells

Type 2 diabetes (T2D) is a major metabolic disease and a key epigenetic risk factor for the development of additional clinical complications. Among them, periodontitis (PD), a severe inflammatory disease ascribable to a dysregulated physiology and composition of the oral microbiota, represents one of the most relevant complications. Periodontitis can impact the structure of the tooth and likely the stem and progenitor cell pool, which actively contributes to the periodontal microenvironment and homeostasis. Modifications of the oral plaque play a key role in the etiopathogenesis of PD caused by T2D. However, to what extent the biology of the progenitor pool is affected has still to be elucidated. In this short report, we aimed to explore the biological effects of oral plaque derived from T2D patients with PD in comparison to non-diabetic patients with PD. Oral plaque samples were isolated from T2D and non-diabetic subjects with PD. Dental pulp stem cells (DPSCs), derived from the premolar tooth, were conditioned for 21 days with oral plaque samples and tested for their clonogenic ability. Cultures were also induced to differentiate towards the osteogenic lineage, and ALP and osteocalcin gene expression levels were evaluated by real-time qPCR. Results have shown that the number of clones generated by DPSCs exposed to T2D oral plaque was significantly lower compared to controls (ctl). The multivariate analysis confirmed that the decreased clonogenesis was significantly correlated only with T2D diagnosis. Moreover, the effect of T2D oral plaque was specific to DPSCs. Indicators of osteogenic differentiation were not significantly affected. This study provides a new biological insight into the effects ascribable to T2D in PD

Influence of Egr-1 in cardiac tissue-derived mesenchymal stem cells in response to glucose variations

Mesenchymal stem cells (MSCs) represent a promising cell population for cell therapy and regenerative medicine applications. However, how variations in glucose are perceived by MSC pool is still unclear. Since, glucose metabolism is cell type and tissue dependent, this must be considered when MSCs are derived from alternative sources such as the heart. The zinc finger transcription factor Egr-1 is an important early response gene, likely to play a key role in the glucose-induced response. Our aim was to investigate how short-term changes in in vitro glucose concentrations affect multipotent cardiac tissue-derived MSCs (cMSCs) in a mouse model of Egr-1 KO (Egr-1-/-). Results showed that loss of Egr-1 does not significantly influence cMSC proliferation. In contrast, responses to glucose variations were observed in wt but not in Egr-1 -/- cMSCs by clonogenic assay. Phenotype analysis by RT-PCR showed that cMSCs Egr-1-/- lost the ability to regulate the glucose transporters GLUT-1 and GLUT-4 and, as expected, the Egr-1 target genes VEGF, TGFβ-1, and p300. Acetylated protein levels of H3 histone were impaired in Egr-1-/- compared to wt cMSCs. We propose that Egr-1 acts as immediate glucose biological sensor in cMSCs after a short period of stimuli, likely inducing epigenetic modifications. © 2014 Daniela Bastianelli et al

Platelet lysate-derived neuropeptide y influences migration and angiogenesis of human adipose tissue-derived stromal cells

Neuropeptide Y (NPY), a powerful neurotransmitter of the central nervous system, is a key regulator of angiogenesis and biology of adipose depots. Intriguingly, its peripheral vascular and angiogenic powerful activity is strictly associated to platelets, which are source of clinical hemoderivates, such as platelet lysate (PL), routinely employed in several clinical applications as wound healing, and to preserve ex vivo the progenitor properties of the adipose stromal cells pool. So far, the presence of NPY in PL and its biological effects on the adipose stromal cell fraction (ASCs) have never been investigated. Here, we aimed to identify endogenous sources of NPY such as PL-based preparations and to investigate which biological properties PL-derived NPY is able to exert on ASCs. The results show that PL contains a high amount of NPY, which is in part also excreted by ASCs when stimulated with PL. The protein levels of the three main NPY subtype receptors (Y1, Y2, Y5) are unaltered by stimulation of ASCs with PL, but their inhibition through selective pharmacological antagonists, considerably enhances migration, and a parallel reduction of angiogenic features of ASCs including decrease in VEGF mRNA and intracellular calcium levels, both downstream targets of NPY. The expression of VEGF and NPY is enhanced within the sites of neovascularisation of difficult wounds in patients after treatment with leuco-platelet concentrates. Our data highlight the presence of NPY in PL preparations and its peripheral effects on adipose progenitors

A review of the molecular mechanisms underlying the development and progression of cardiac remodeling

Pathological molecular mechanisms involved in myocardial remodeling contribute to alter the existing structure of the heart, leading to cardiac dysfunction. Among the complex signaling network that characterizes myocardial remodeling, the distinct processes are myocyte loss, cardiac hypertrophy, alteration of extracellular matrix homeostasis, fibrosis, defective autophagy, metabolic abnormalities, and mitochondrial dysfunction. Several pathophysiological stimuli, such as pressure and volume overload, trigger the remodeling cascade, a process that initially confers protection to the heart as a compensatory mechanism. Yet chronic inflammation after myocardial infarction also leads to cardiac remodeling that, when prolonged, leads to heart failure progression. Here we review the molecular pathways involved in cardiac remodeling, with particular emphasis on those associated with myocardial infarction. A better understanding of cell signaling involved in cardiac remodeling may support the development of new therapeutic strategies towards the treatment of heart failure and reduction of cardiac complications. We will also discuss data derived from gene therapy approaches for modulating key mediators of cardiac remodeling

Remote adipose tissue-derived stromal cells of patients with lung adenocarcinoma generate a similar malignant microenvironment of the lung stromal counterpart

Cancer alters both local and distant tissue by influencing the microenvironment. In this regard, the interplay with the stromal fraction is considered critical as this latter can either foster or hamper the progression of the disease. Accordingly, the modality by which tumors may alter distant niches of stromal cells is still unclear, especially at early stages. In this short report, we attempt to better understand the biology of this cross-talk. In our “autologous stromal experimental setting,” we found that remote adipose tissue-derived mesenchymal stem cells (mediastinal AMSC) obtained from patients with lung adenocarcinoma sustain proliferation and clonogenic ability of A549 and human primary lung adenocarcinoma cells similarly to the autologous stromal lung counterpart (LMSC). This effect is not observed in lung benign diseases such as the hamartochondroma. This finding was validated by conditioning benign AMSC with supernatants from LAC for up to 21 days. The new reconditioned media of the stromal fraction so obtained, was able to increase cell proliferation of A549 cells at 14 and 21 days similar to that derived from AMSC of patients with lung adenocarcinoma. The secretome generated by remote AMSC revealed overlapping to the corresponding malignant microenvironment of the autologous local LMSC. Among the plethora of 80 soluble factors analyzed by arrays, a small pool of 5 upregulated molecules including IL1-β, IL-3, MCP-1, TNF-α, and EGF, was commonly shared by both malignant-like autologous A- and L-MSC derived microenvironments vs those benign. The bioinformatics analysis revealed that these proteins were strictly and functionally interconnected to lung fibrosis and proinflammation and that miR-126, 101, 486, and let-7-g were their main targets. Accordingly, we found that in lung cancer tissues and blood samples from the same set of patients here employed, miR-126 and miR-486 displayed the highest expression levels in tissue and blood, respectively. When the miR-126-3p was silenced in A549 treated with AMSC-derived conditioned media from patients with lung adenocarcinoma, cell proliferation decreased compared to control media

Inactivation of the glutathione peroxidase GPx4 by the ferroptosis-inducing molecule RSL3 requires the adaptor protein 14-3-3 epsilon

RSL3, a drug candidate prototype for cancer chemotherapy, triggers ferroptosis by inactivating GPx4. Here we report the purification of the protein indispensable for GPx4 inactivation by RSL3. MS analysis reveals 14-3-3 isoforms as candidates and recombinant human 14-3-3epsilon confirms the identification. The function of 14-3-3\uf065 is redox-regulated. Moreover, overexpression and silencing of the gene coding for 14-3-3\uf065 consistently control the inactivation of GPx4 by RSL3. The interaction of GPx4 with a redox-regulated adaptor protein, operating in cell signalling, further contributes to frame it within redox-regulated pathways of cell survival and death and opens new therapeutic perspectives

The impact of environmental factors in influencing epigenetics related to oxidative states in the cardiovascular system

Oxidative states exert a significant influence on a wide range of biological and molecular processes and functions. When their balance is shifted towards enhanced amounts of free radicals, pathological phenomena can occur, as the generation of reactive oxygen species (ROS) in tissue microenvironment or in the systemic circulation can be detrimental. Epidemic chronic diseases of western societies, such as cardiovascular disease, obesity, and diabetes correlate with the imbalance of redox homeostasis. Current advances in our understanding of epigenetics have revealed a parallel scenario showing the influence of oxidative stress as a major regulator of epigenetic gene regulation via modification of DNA methylation, histones, and microRNAs. This has provided both the biological link and a potential molecular explanation between oxidative stress and cardiovascular/metabolic phenomena. Accordingly, in this review, we will provide current insights on the physiological and pathological impact of changes in oxidative states on cardiovascular disorders, by specifically focusing on the influence of epigenetic regulation. A special emphasis will highlight the effect on epigenetic regulation of human's current life habits, external and environmental factors, including food intake, tobacco, air pollution, and antioxidant-based approaches. Additionally, the strategy to quantify oxidative states in humans in order to determine which biological marker could best match a subject's profile will be discussed