Anti-inflammatory activity of Wnt signaling in enteric nervous system: in vitro preliminary evidences in rat primary cultures

Background: In the last years, Wnt signaling was demonstrated to regulate inflammatory processes. In particular, an increased expression of Wnts and Frizzled receptors was reported in inflammatory bowel disease (IBD) and ulcerative colitis to exert both anti- and pro-inflammatory functions regulating the intestinal activated nuclear factor \u3baB (NF-\u3baB), TNFa release, and IL10 expression. Methods: To investigate the role of Wnt pathway in the response of the enteric nervous system (ENS) to inflammation, neurons and glial cells from rat myenteric plexus were treated with exogenous Wnt3a and/or LPS with or without supporting neurotrophic factors such as basic fibroblast growth factor (bFGF), epithelial growth factor (EGF), and glial cell-derived neurotrophic factor (GDNF). The immunophenotypical characterization by flow cytometry and the protein and gene expression analysis by qPCR and Western blotting were carried out. Results: Flow cytometry and immunofluorescence staining evidenced that enteric neurons coexpressed Frizzled 9 and toll-like receptor 4 (TLR4) while glial cells were immunoreactive to TLR4 and Wnt3a suggesting that canonical Wnt signaling is active in ENS. Conclusions: The results of this study suggested the existence of neuronal surveillance through FZD9 and Wnt3a in enteric myenteric plexus. Moreover, experimental evidences were provided to clarify the correlation among soluble trophic factors, Wnt signaling, and anti-inflammatory protection of ENS

Breast cancer cells grown on hyaluronic acid-based scaffolds as 3D in vitro model for electroporation

Nowadays, electroporation (EP) represents a promising method for the intracellular delivery of anticancer drugs. To setting up the process, the EP efficiency is usually evaluated by using cell suspension and adherent cell cultures that are not representative of the in vivo conditions. Indeed, cells are surrounded by extracellular matrix (ECM) whose composition and physical characteristics are different for each tissue. So, various three-dimensional (3D) in vitro models, such as spheroids and hydrogel-based cultures, have been proposed to mimic the tumour microenvironment. Herein, a 3D breast cancer in vitro model has been proposed. HCC1954 cells were seeded on crosslinked and lyophilized matrices composed of hyaluronic acid (HA) and ionic complementary self-assembling peptides (SAPs) already known to provide a fibrous structure mimicking collagen network. Herein, SAPs were functionalized with laminin derived IKVAV adhesion motif. Cultures were characterized by spheroids surrounded by ECM produced by cancer cells as demonstrated by collagen1a1 and laminin B1 transcripts. EP was carried out on both 2D and 3D cultures: a sequence of 8 voltage pulses at 5 kHz with different amplitude was applied using a plate electrode. Cell sensitivity to EP seemed to be modulated by the presence of ECM and the different cell organization. Indeed, cells cultured on HA-IKVAV were more sensitive than those treated in 2D and HA cultures, in terms of both cell membrane permeabilization and viability. Collectively, our results suggest that HA-IKVAV cultures may represent an interesting model for EP studies. Further studies will be needed to elucidate the influence of ECM composition on EP efficiency

Nanopatterned acellular valve conduits drive the commitment of blood-derived multipotent cells

Considerable progress has been made in recent years toward elucidating the correlation among nanoscale topography, mechanical properties, and biological behavior of cardiac valve substitutes. Porcine TriCol scaffolds are promising valve tissue engineering matrices with demonstrated self-repopulation potentiality. In order to define an in vitro model for investigating the influence of extracellular matrix signaling on the growth pattern of colonizing blood-derived cells, we cultured circulating multipotent cells (CMC) on acellular aortic (AVL) and pulmonary (PVL) valve conduits prepared with TriCol method and under no-flow condition. Isolated by our group from Vietnamese pigs before heart valve prosthetic implantation, porcine CMC revealed high proliferative abilities, three-lineage differentiative potential, and distinct hematopoietic/endothelial and mesenchymal properties. Their interaction with valve extracellular matrix nanostructures boosted differential messenger RNA expression pattern and morphologic features on AVL compared to PVL, while promoting on both matrices the commitment to valvular and endothelial cell-like phenotypes. Based on their origin from peripheral blood, porcine CMC are hypothesized in vivo to exert a pivotal role to homeostatically replenish valve cells and contribute to hetero- or allograft colonization. Furthermore, due to their high responsivity to extracellular matrix nanostructure signaling, porcine CMC could be useful for a preliminary evaluation of heart valve prosthetic functionality

Neurological, psychological, and cognitive disorders in patients with chronic kidney disease on conservative and replacement therapy

Chronic kidney disease (CKD) is a highly prevalent condition in the world. Neurological, psychological, and cognitive disorders, related to CKD, could contribute to the morbidity, mortality, and poor quality of life of these patients. The aim of this study was to assess the neurological, psychological, and cognitive imbalance in patients with CKD on conservative and replacement therapy. Seventy-four clinically stable patients affected by CKD on conservative therapy, replacement therapy (hemodialysis (HD), peritoneal dialysis (PD)), or with kidney transplantation (KT) and 25 healthy controls (HC), matched for age and sex were enrolled. Clinical, laboratory, and instrumental examinations, as renal function, inflammation and mineral metabolism indexes, electroencephalogram (EEG), psychological (MMPI-2, Sat P), and cognitive tests (neuropsychological tests, NPZ5) were carried out. The results showed a significant differences in the absolute and relative power of delta band and relative power of theta band of EEG (P=0.008, P<0.001, P=0.051), a positive correlation between relative power of delta band and C-reactive protein (CRP) (P< 0.001) and a negative correlation between estimated glomerular filtration rate (eGFR) (P<0.001) and 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3) (P<0.001), in all the samples. Qualitative analysis of EEG showed alterations of Grade 2 (according to Parsons-Smith classification) in patients on conservative therapy, and Grade 2-3 in KT patients. The scales of MMPI-2 hysteria and paranoia, are significantly correlated with creatinine, eGFR, serum nitrogen, CRP, 1,25-(OH)2D3, intact parathyroid hormone (iPTH), phosphorus, and cynical and hysterical personality, are correlated with higher relative power of delta (P=0.016) and theta band (P= 0.016). Moreover, all NPZ5 scores showed a significant difference between the means of nephropathic patients and the means of the HC, and a positive correlation with eGFR, serum nitrogen, CRP, iPTH, and vitamin D. In CKD patients, simple and noninvasive instruments, as EEG, and cognitive-psychological tests, should be performed and careful and constant monitoring of renal risk factors, probably involved in neuropsychological complications (inflammation, disorders of mineral metabolism, electrolyte disorders, etc.), should be carried out. Early identification and adequate therapy of neuropsychological, and cognitive disorders, might enable a better quality of life and a major compliance with a probable reduction in the healthcare costs

Caratterizzazione e plasticità differenziativa di popolazioni cellulari staminali isolate da sangue periferico

Stem cells are primitive undifferentiated cells with unlimited proliferative capacity and defined differentiative abilities under specific stimula. Over the last decade, great interest has been addressed versus the clinical use of adult stem cells that, in comparison to embryonic stem cells, do not involve the destruction of embryos to be obtained and therefore do not induce any ethical problem. Adult stem cells are mostly multipotent and are present within niches in adipose tissue, bone marrow and skin of adults and children. Experimental studies have shown that multipotent stem cells are present in animal and human peripheral blood and their potentialities make them ideal candidate for clinical programs of cell therapy to restore structural or functional tissue defects that are difficult to be solved with traditional medical therapies. It was also shown that, using in vitro defined culture conditions, they can be modified acquiring cardiomyocyte phenotype. After 24h exposure with DNA demethylating chemical 5-azacitidine (5-AZA), the commonest strategy to induce the in vitro cardiac differentiation, MSC demonstrated a cardiomyocyte-like ultrastructure, including typical sarcomeres, a centrally positioned nucleus and atrial granules. The expression of contractile protein genes for myosin heavy chain, myosin light chain and α-actin, confirmed them to be similar to the foetal ventricular cardiomyocytes. In the last decade, the development of the heart valve tissue engineering provided promises to obtain fully compatible grafts able, after transplantation, to be in vivo remodelled. Researchers have demonstrated the efficacy of acellular and natural matrices to repair muscle, bone and trachea. Some of the most widely used techniques have involved detergents such as sodium dodecylsulphate (SDS), sodium deoxycholate (SD), sodium cholate (SC), Triton X–100 (TX) and trypsin enzyme. TRICOL, a technique employing TX, sodium cholate (SC), hypo- and hyper-tonic solutions and Benzonase has been developed by Spina et al. to obtain porcine aortic and pulmonary valves. Acellular matrices have been tested in combination or not with differentiated tissue-specific cells (endothelial and/or smooth muscle cells), autologous or allogenic stem cells. Seeding peripheral blood-derived autologous cells on acellular matrices could optimize the in vivo functionality of the implant to its final complete integration. The aim of this thesis has been the isolation of circulating cells from peripheral blood to be used in tissue engineering strategies. The study envolved minipig as animal model. CD44high, CD106+, CD90low, SLA-DRlow, CD45-, CD34-, CXCR4- (CSP) fibroblastic cells have been isolated using the strategy by adherence to plastic. The long term expansion has been performed for 39 passages and demonstrated that CSP phenotype and morphology are highly stable, as cytometrical study and microscopical analysis demonstrated on subcultures VIII, XXIV and XXXIX. Moreover, the specific induction with adipogenic, osteogenic and cardiomyogenic media revealed that CSP cells are able to specifically respond to the induction stimula, accumulating lipids within cytoplasm, producing mineralized extracellular matrix and expressing cardiomyogenic markers such as phospholamban, alpha actin muscle specific and troponin T. In order to test the clinical potentialities of CD44high, CD106+, CD90low, SLA-DRlow, CD45-, CD34-, CXCR4- circulating cells, in cardiological field, the seeding of CSP cells has been performed on acellular porcine valves (VD) (aortic and pulmonary) as scaffolds. The cellular adhesion has been studied by SEM at different time points (7, 14, 28, 35 and 42 days). The morphological study by SEM demonstrated that CSP cells colonized the seeding surface and organized themselves into continuous monolayers. The histological study by immunofluorescence showed the presence of elastin and collagen (IV and I) whereas immunohystochemistry demonstrated the positivity of cells for alpha actin.Le cellule staminali sono cellule primitive indifferenziate, dotate di capacità proliferativa illimitata e in grado di differenziare in cellule specializzate in presenza di stimoli specifici. Nell’ultimo decennio, grande interesse è stato rivolto all’impiego in campo medico di cellule staminali adulte che, rispetto alle cellule staminali embrionali totipotenti, non comportano la distruzione dell’embrione per il loro ottenimento e pertanto non sollevano alcun problema di carattere etico. Le cellule staminali adulte sono cellule prevalentemente multipotenti, identificate in nicchie a livello del tessuto adiposo, del midollo osseo e della cute di individui adulti e bambini. Studi sperimentali hanno evidenziato che nel sangue periferico animale e umano sono presenti cellule ad alta capacità plastica e che dunque potrebbero essere utilizzate in clinica per lo sviluppo di programmi di terapia cellulare ed il recupero di danni tessutali refrattari alle terapie mediche classiche. Le cellule staminali mesenchimali (MSC) hanno dimostrato di essere idonee all’impiego in vivo per lo sviluppo di programmi di terapia cellulare. È stato inoltre dimostrato che, in definite condizioni di coltura, le cellule MSC possono acquisire un fenotipo simil-cardiomiocitario. La strategia più comune utilizzata per indurre il loro differenziamento cardiomiogenico in vitro è il trattamento per 24 ore con la 5-azacitidina (5-AZA), un agente chimico demetilante del DNA. Dopo induzione, le cellule mostrano una morfologia miotubulare, caratterizzata dalla presenza di unità sarcomeriche, un nucleo centrale e tipici granuli atriali. Similmente ai cardiomiociti fetali ventricolari, esse esprimono proteine contrattili, quali l’α-actina, la catena pesante (MHC) e leggera della miosina (MLC). Nel corso degli ultimi dieci anni, lo sviluppo dell’ingegneria tessutale delle valvole cardiache ha aperto un nuovo orizzonte verso l’utilizzo di un costrutto biocompatibile in grado di essere rimodellato dopo l’impianto. Numerosi studi hanno messo in luce l’efficacia delle matrici naturali acellulari per il recupero di danni tessutali a livello muscolare, osseo e tracheale. I metodi sperimentati per la decellularizzazione di valvole aortiche e polmonari hanno previsto l’uso di detergenti quali il sodio dodecilsolfato (SDS), il sodio deossicolato (SD), il sodio colato (SC), il Triton X–100 (TX) e l’enzima tripsina. Il trattamento TRICOL, che comprende l’uso di TX, sodio colato (SC), soluzioni ipo- ed iper-toniche e Benzonase, è stato sviluppato da Spina e collaboratori per la preparazione di valvole porcine aortiche e polmonari. L’ingegnerizzazione della matrice con cellule autologhe isolate da sangue periferico potrebbe ottimizzare l’attecchimento dell’impianto e consentire il suo mantenimento in sede fino a completa integrazione. Lo scopo della tesi è stato isolare cellule circolanti multipotenti da sangue periferico, idonee ad applicazioni di ingegneria tessutale. Lo studio è stato eseguito utilizzando il modello animale minipig. Sono state isolate popolazioni fibroblastoidi mediante la tecnica di adesione alla plastica, aventi fenotipo CD44high, CD106+, CD90low, SLA-DRlow, CD45-, CD34-, CXCR4- (CSP). L’espansione a lungo termine, eseguita per 39 passaggi, ha dimostrato che le popolazioni CSP sono altamente stabili da un punto di vista fenotipico e morfologico, come dimostrato mediante analisi citometrica e di microscopia ottica su subcolture intermedie VIII, XXIV e XXXIX. Inoltre, la specifica induzione con terreni differenziativi in senso adipogenico, osteogenico e cardiomiogenico ha dimostrato che le cellule CSP sono cellule plastiche, che rispondono agli stimoli in modo specifico accumulando trigliceridi, producendo matrice extracellulare mineralizzata ed esprimendo marcatori cardiomiogenici quali il fosfolambano, l’alfa-actina muscolo specifica e la troponina cardiaca T. Allo scopo di valutare le potenzialità di impiego in campo cardiologico di popolazioni circolanti con immunofenotipo CD44high, CD106+, CD90low, SLA-DRlow, CD45-, CD34-, CXCR4-, è stata oggetto di studio la crescita delle cellule CSP su matrici valvolari decellularizzate (VD) aortiche e polmonari. La valutazione dell’adesione è stata eseguita mediante analisi SEM a differenti intervalli di tempo (7, 14, 28, 35 e 42 giorni). Lo studio morfologico mediante analisi SEM ha dimostrato la colonizzazione della superficie di semina da parte delle cellule CSP e la loro organizzazione in monostrati continui. Lo studio istologico mediante immunofluorescenza ha evidenziato sui campioni la presenza di elastina e di collagene (IV e I) mentre lo studio immunoistochimico ha dimostrato la positività delle cellule per l’alfa-actina cellulare scheletrica

How Blue Light Activates Furocoumarin Derivatives Triggering Tumor Cell Apoptosis

Furocoumarins comprise natural and synthetic compounds: linear molecules, so called psoralens, and the angular ones, the angelicins. The photobiological effects of furocoumarins plus UVA are mainly related to their capacity to bind DNA and form monoadducts (MAs) and interstrand crosslinks (XLs), mainly with pyrimidine bases. Furthermore, furocoumarins produce ROS that impair cellular functions through lipid peroxidation, oxidation of guanine and strand breaks in nucleic acids, oxidation of proteins and inactivation of enzymes. It is known that the combination of 8-MOP and UVA radiation causes apoptosis of treated leucocytes and may cause preferential apoptosis of activated or abnormal T cells. Moreover, these apoptotic cells may promote immune tolerance, production of antigen-specific regulatory lymphocytes (CD4/8 T, B) and rebalance of immune system. Even though furocoumarins possess high chemotherapeutic potency under UVA and lack toxicity in the dark, genotoxicity, mutagenicity and skin phototoxicity have been observed. 8-MOP was found to photoreact under blue light (BL), leading to less mutagenic lesions in the DNA, that is preferentially MAs over XLs. Furthermore, cells treated with 419 nm light resumed normal growth rates faster than cells which received the same UVA dose

4,6,4′-Trimethylangelicin Photoactivated by Blue Light Might Represent an Interesting Option for Photochemotherapy of Non-Invasive Bladder Carcinoma: An In Vitro Study on T24 Cells

Photodynamic therapy (PDT) is frequently used to treat non-muscle invasive bladder cancer due its low toxicity and high selectivity. Since recurrence often occurs, alternative approaches and/or designs of combined therapies to improve PDT effectiveness are needed. This work aimed to evaluate the cytotoxicity of 4,6,4&prime;-trimethylangelicin (TMA) photoactivated by blue light (BL) on human bladder cancer T24 cells and investigate the mechanisms underlying its biological effects. TMA/BL exerted antiproliferative activity through the induction of apoptosis without genotoxicity, as demonstrated by the expression levels of phospho-H2AX, an indicator of DNA double-stranded breaks. It also modulated the Wnt canonical signal pathway by increasing the phospho-&beta;-catenin and decreasing the nuclear levels of &beta;-catenin. The inhibition of this pathway was due to the modulation of the GSK3&beta; phosphorylation state (Tyr 216) that induces a proteasomal degradation of &beta;-catenin. Indeed, a partial recovery of nuclear &beta;-catenin expression and reduction of its phosphorylated form after treatment with LiCl were detected. As demonstrated by RT-PCR and cytofluorimetric analysis, TMA/BL also decreased the expression of CD44v6, a marker of cancer stem cells. Taken together, our data suggest that TMA photoactivated by BL may represent an interesting option for the photochemotherapy of noninvasive bladder carcinomas, since this treatment is able to inhibit key pathways for tumour growth and progression in the absence of genotoxic effects

4,6,4′-Trimethylangelicin Photoactivated by Blue Light Might Represent an Interesting Option for Photochemotherapy of Non-Invasive Bladder Carcinoma: An In Vitro Study on T24 Cells

Photodynamic therapy (PDT) is frequently used to treat non-muscle invasive bladder cancer due its low toxicity and high selectivity. Since recurrence often occurs, alternative approaches and/or designs of combined therapies to improve PDT effectiveness are needed. This work aimed to evaluate the cytotoxicity of 4,6,4′-trimethylangelicin (TMA) photoactivated by blue light (BL) on human bladder cancer T24 cells and investigate the mechanisms underlying its biological effects. TMA/BL exerted antiproliferative activity through the induction of apoptosis without genotoxicity, as demonstrated by the expression levels of phospho-H2AX, an indicator of DNA double-stranded breaks. It also modulated the Wnt canonical signal pathway by increasing the phospho-β-catenin and decreasing the nuclear levels of β-catenin. The inhibition of this pathway was due to the modulation of the GSK3β phosphorylation state (Tyr 216) that induces a proteasomal degradation of β-catenin. Indeed, a partial recovery of nuclear β-catenin expression and reduction of its phosphorylated form after treatment with LiCl were detected. As demonstrated by RT-PCR and cytofluorimetric analysis, TMA/BL also decreased the expression of CD44v6, a marker of cancer stem cells. Taken together, our data suggest that TMA photoactivated by BL may represent an interesting option for the photochemotherapy of noninvasive bladder carcinomas, since this treatment is able to inhibit key pathways for tumour growth and progression in the absence of genotoxic effects