Mesenchymal Stem Cells Increase Neo-Angiogenesis and Albumin Production in a Liver Tissue-Engineered Engraftment

The construction of a three-dimensional (3D) liver tissue is limited by many factors; one of them is the lack of vascularization inside the tissue-engineered construct. An engineered liver pocket-scaffold able to increase neo-angiogenesis in vivo could be a solution to overcome these limitations. In this work, a hyaluronan (HA)-based scaffold enriched with human mesenchymal stem cells (hMSCs) and rat hepatocytes was pre-conditioned in a bioreactor system, then implanted into the liver of rats. Angiogenesis and hepatocyte metabolic functions were monitored. The formation of a de novo vascular network within the HA-based scaffold, as well as an improvement in albumin production by the implanted hepatocytes, were detected. The presence of hMSCs in the HA-scaffold increased the concentration of growth factors promoting angiogenesis inside the graft. This event ensured a high blood vessel density, coupled with a support to metabolic functions of hepatocytes. All together, these results highlight the important role played by stem cells in liver tissue-engineered engraftment

ESCLUSIONE ENDOVASCOLARE DI PSEUDOANEURISMI IN FEGATO TRAPIANTATI: VIRTUTE DUCE, COMITE FORTUNA

Scopo: Presentare due casi di trattamento endovascolare di pseudoaneurismi in pazienti sottoposti a trapianto epatico. Materiali e metodi: Gli pseudoaneurismi sono stati identificati incidentalmente in corso di follow-up dopo trapianto epatico ortotopico. Il paziente 1, nel quale era stata confezionata una bilio-digiuno anastomosi, presentava uno pseudoaneurisma pre-anastomotico dell’arteria epatica, con trombosi dell’arteria a valle ed opacizzazione dei rami intraepatici apparentemente attraverso esili circoli collaterali di pertinenza dell’arteria epatica comune e del circolo mesenterico. Il paziente 2 presentava uno pseudoaneurisma intraepatico, probabile conseguenza di biopsia eseguita sul donatore. Risultati: In entrambi i casi lo pseudoaneurisma è stato escluso dal circolo sistemico edil graft è stato preservato. Nel paziente 1 una delle spirali metalliche utilizzate per l’esclusione dello pseudoaneurisma con tecnica “sandwich“ si è dislocata trombizzando l’arteria epatica comune prossimale; fortunatamente, grazie a verosimili fenomeni di neoangiogenesi indotti dalla presenza della bilio-digiuno anastomosi, le branche arteriose intraepatiche permanevano pervie, anche in corso di follow- up. Nel paziente 2 si è scelto di utilizzare la tecnica “jailing“, con posizionamento di spirali a rilascio controllato attraverso le maglie di uno stent a celle aperte posizionato nel lume dell’arteria coinvolta dallo pseudoaneurisma; nonostante una parziale occlusione dello stent, le diramazioni a valle permanevano pervie in corso di follow-up. Conclusioni: Nel fegato trapiantato il circolo arterioso è importante quanto quello portale. Il trattamento endovascolare in tali casi è una procedura ad elevato rischio, poichè l’embolizzazione non target può portare a perdita di parenchima con malfunzionamento del graft

In vitro effects of interleukin (IL)-1 beta inhibition on the epithelial-to-mesenchymal transition (EMT) of renal tubular and hepatic stellate cells

BACKGROUND: The epithelial to mesenchymal transition (EMT) is a multi-factorial biological mechanism involved in renal and hepatic fibrosis and the IL-1 beta has been assumed as a mediator of this process although data are not exhaustive. Therefore, the aim of our study was to evaluate the role of this cytokine in the EMT of renal proximal tubular epithelial cells (HK-2) and stellate cells (LX-2) and the protective/anti-fibrotic effect of its inhibition by Canakinumab (a specific human monoclonal antibody targeted against IL-1beta). METHODS: Both cell types were treated with IL-1 beta (10 ng/ml) for 6 and 24 h with and without Canakinumab (5 \u3bcg/ml). As control we used TGF-beta (10 ng/ml). Expression of EMT markers (vimentin, alpha-SMA, fibronectin) were evaluated through western blotting and immunofluorescence. Genes expression for matrix metalloproteinases (MMP)-2 was measured by Real-Time PCR and enzymatic activity by zymography. Cellular motility was assessed by scratch assay. RESULTS: IL-1 beta induced a significant up-regulation of EMT markers in both cell types and increased the MMP-2 protein expression and enzymatic activity, similarly to TGF-beta. Moreover, IL-1 beta induced a higher rate of motility in HK-2. Canakinumab prevented all these modifications in both cell types. CONCLUSIONS: Our results clearly demonstrate the role of IL-1 beta in the EMT of renal/stellate cells and it underlines, for the first time, the therapeutic potential of its specific inhibition on the prevention/minimization of organ fibrosis

Sirolimus and Everolimus Pathway: Reviewing Candidate Genes Influencing Their Intracellular Effects

Sirolimus (SRL) and everolimus (EVR) are mammalian targets of rapamycin inhibitors (mTOR-I) largely employed in renal transplantation and oncology as immunosuppressive/antiproliferative agents. SRL was the first mTOR-I produced by the bacterium Streptomyces hygroscopicus and approved for several medical purposes. EVR, derived from SRL, contains a 2-hydroxy-ethyl chain in the 40th position that makes the drug more hydrophilic than SRL and increases oral bioavailability. Their main mechanism of action is the inhibition of the mTOR complex 1 and the regulation of factors involved in a several crucial cellular functions including: protein synthesis, regulation of angiogenesis, lipid biosynthesis, mitochondrial biogenesis and function, cell cycle, and autophagy. Most of the proteins/enzymes belonging to the aforementioned biological processes are encoded by numerous and tightly regulated genes. However, at the moment, the polygenic influence on SRL/EVR cellular effects is still not completely defined, and its comprehension represents a key challenge for researchers. Therefore, to obtain a complete picture of the cellular network connected to SRL/EVR, we decided to review major evidences available in the literature regarding the genetic influence on mTOR-I biology/pharmacology and to build, for the first time, a useful and specific "SRL/EVR genes-focused pathway", possibly employable as a starting point for future in-depth research projects

Epithelial to mesenchymal transition in the liver field: the double face of Everolimus in vitro.

Everolimus (EVE), a mammalian target of rapamycin inhibitor, has been proposed as liver transplant immunosuppressive drug, gaining wide interest also for the treatment of cancer. Although an appropriate tolerance, it may induce several adverse effects, such as fibro-interstitial pneumonitis due to the acquisition of activated myofibroblasts. The exact molecular mechanism associated with epithelial to mesenchymal transition (EMT) may be crucial also in the liver context. This work examines the role and the molecular mediators of EMT in hepatic stellate cell (HSC) and human liver cancer cells (HepG2) and the potential role of EVE to maintain the epithelial phenotype rather than to act as a potential initiators of EMT.Real time-PCR and western blot have been used to assess the capability of EVE at low-therapeutic (10 nM) and high (100 nM) dose to induce an in vitro EMT in HSC and HepG2.Biomolecular experiments demonstrated that low concentration of EVE (10 nM) did not modify the gene expression of alpha-smooth muscle actin (α-SMA), Vimentin (VIM), Fibronectin (FN) in both HSC and HepG2 cells, whereas EVE at 100 nM induced a significant over-expression of all the three above-mentioned genes and an increment of α-SMA and FN protein levels. Additionally, 100 nM of EVE induced a significant phosphorylation of AKT and an up-regulation of TGF-β expression in HSC and HepG2 cells.Our data, although obtained in an in vitro model, revealed, for the first time, that high concentration of EVE may induce EMT in liver cells confirming previous published evidences obtained in renal cells. Additionally, they suggested that mTOR-I should be administered at the lowest dose able to maximize their important and specific therapeutic properties minimizing or avoiding fibrosis-related adverse effects.In summary, if confirmed by additional studies, our results could be useful for researchers to standardize new therapeutic immunosuppressive and anticancer drugs protocols

Correction to: Effects of immune suppression for transplantation on inflammatory colorectal cancer progression (Oncogenesis, (2018), 7, 6, (46), 10.1038/s41389-018-0055-5)

At the time of publication, the html version of this paper contained an error; the authors Imerio Angriman and Lucrezia Furian were not tagged as equally contributing authors. This has now been fixed in the html version of the paper, the PDF was correct at the time of publication. Erratum for Effects of immune suppression for transplantation on inflammatory colorectal cancer progression. [Oncogenesis. 2018

LC3B and ph-S6K are both expressed in epithelioid and classic renal angiomyolipoma: a rationale tissue-based evidence for combining use of autophagic and mTOR targeted drugs

Background: Targeted drugs to the autophagy processes are emerging in clinical trials. The aim of this work is to assess the magnitude of autophagic expression in renal angiomyolipoma. Methods: Fourteen cases of renal angiomyolipoma were recruited. Anti-LC3B-II and anti-phospho-S6K were detected by Western blot analysis. For immunohistochemical staining, sections were stained with the antibodies LC3B-II and cathepsin-K. LC3B-II was also analyzed by immunofluorescence. We have also carried out electron microscopy analysis on tumor cells. Results: 13 classic and 1 epithelioid renal angiomyolipoma were recruited. The Western-blot LC3B-II analysis shows increasing in protein expression in all cases, however quantitative protein expression ranged from 1 to 15 (mean 5). The autophagosome protein LC3B-I also significantly increased in all tumor extraction. The expression of LC3B-II protein was confirmed in tumoral samples by immunofluorescence. The lysosomal marker cathepsin-K was observed by immunohistochemistry on all tumours. The Western-blot ph-S6K analysis showed significant protein overexpression along all cases after evaluation of the quantitative S6K/Ponceaus ratio. In 6/14 (52%) the expression was high, with a quantitative increase of 653 fold induction in 4 angiomyolipoma compared to normal tissue. At electron microscopy, cancer cells evidenced round or oval electron-dense granules associated with membranes and granules with double membrane. Conclusion: Both autophagic LC3B-II and ph-S6K molecules are over-represented in both epithelioid and classic renal angiomyolipoma and a combined use of inhibitors to the autophagic and mTOR processes may be designed in clinical trials, when enrolling patients affected by tumours in tuberous sclerosis or angiomyolipoma at risk of bledding

Realizzazione multistep di neo-costrutti epatici con apporto vascolare attraverso tecniche di tissue engineering

INTRODUCTION: This multistep work has investigated tissue engineering of the liver as approach for treating end-stage liver diseases by different projects. Engineering of hepatic tissue based on primary hepatocytes offers new perspectives in this field. However, generation of thick, 3D liver tissue has been limited by the lack of vasculature in the engineered constructs; hepatocyte survival is transient if limited by insufficient vascular-network formation. To overcome this limitation and establish a vascularized construct, we firstly worked on designing a novel microfluidic-based bilayer device with a discrete parenchymal chamber modeled upon hepatic organ architecture. The design enables the device concept to serve as both a platform technology for drug discovery and toxicity, and for the continuing development of an improved liver-assist device. Thereafter the designed device has been moved to an in vivo animal model (project 2), evaluating the liver-assist device platform with a microfluidic-modeled vascular network in a femoral arteriovenous shunt model in rats. On this basis, we proceeded with efficiently differentiating precursor liver cells into mature cells within 3D bioreactor systems in different culture conditions. Combined and distinct methods have been tested to enhance the in vitro differentiation of liver precursor cells (project 3). Finally, new smart 3D scaffolds cultured with a mixed population of hepatocytes and mesenchymal stem cells have been implanted in vivo (project 4), after adequate preconditioning in the previous bioreactor system, to induce angiogenesis processes. These different approaches can be efficiently put together for the final realization of an in vivo bio-artificial liver construct. METHODS: Project 1: 18 assembled devices with continuous flow have been tested for the capability of transporting of metabolites and small proteins while protecting an adjacent cell culture from the effects of shear stress. Devices were seeded with HepG2/C3A and primary hepatocytes in different culture conditions to test the final capability of the system to maintain an efficient dynamic culture. Project 2: 16 devices (assembled in project 1) with rat primary hepatocytes and 12 with human HepG2/C3A cells were tested in athymic rats in a femoral arteriovenous shunt model. Several parenchymal tube configurations were evaluated for pressure profile and cell survival. The blood flow pattern and perfusion status of the devices was examined by laser Doppler scanning. Cell viability and serum protein secretion functions were assessed. Project 3: Pluripotent Human Liver Stem Cells (HLSCs) were seeded onto 18 3D "smart scaffolds" composed by a biocompatible collagen-sponge. The scaffold was connected to a novel perfusion bioreactor able to ensure long-term uniform flow of medium through the material sponges; stem cells medium and co-culture with hepatic stellate cells (HSCs) were added in different conditions. Tissue engineering strategies based on the co-cultivation of HLSCs with hepatic stellate cells (ITO) and with several combinations of medium were applied. Morphological and functional assays were performed at day 3, 5 and 7 of the in vitro perfusion condition Project 4: 12 scaffolds composed by 3D hyaluronan-derivative [a benzyl ester of hyaluronan (HYAFF®), seeded with a population of human mesenchymal stem cells (HMSCs) and hepatocytes in different ratio, were firstly cultured in the previous bioreactor system to induce a preconditioning stimulus for proliferation and regeneration. The scaffolds were then implanted onto the omentum of 12 nude rats and then rolled into a 3-D pocket structure. Morphological assays were performed to test the neoangiogenesis properties of HMSCs and establish hepatocytes viability after 7 days. RESULTS: Project 1: The assembled device was able to sustain both human hepatoma cells and primary rat hepatocytes by continuous in vitro perfusion of medium, allowing proliferation and maintaining hepatic functions such as serum protein synthesis and metabolism. The mathematical model estimated the best flow rate for perfused cultures lasting up to 14 days. Project 2: The testing in femoral arteriovenous shunt model was successfully established in all animals. Blood flow was homogeneous through the vascular bed and replicated native flow patterns. Survival of seeded liver cells was highly dependent on parenchymal chamber pressures. The tube configuration that generated the lowest pressure supported excellent cell survival and function. Project 3: The hepatic differentiation of HLSCs from adult liver has been improved in 3D collagen scaffolds, confirmed by morphological and functional assays; the flow of perfusion medium (assured by the bioreactor system) enabled the in vitro organization of the cells into liver clusters even in the deeper levels of the sponge. This preliminary experiment has shown that collagen sponge and dynamic in vitro condition not only promote formation of cellular bodies of HLSCs but also enhance a more rapidly functional differentiation into a mature hepatic population. Project 4: The final ongoing project moved to the in vivo model. After a preconditioning period in the bioreactor assembled in project 3, cultured HYAFF®) 3D scaffold (with hepatocytes plus HMSCs or hepatocytes alone) have been implanted in rats. New tissues consisting of neo-angiogenesis’ clusters organized in vessel-like structures formed by 1 week, and the hepatocyte mass survived during all the study-time. Vascular structures, identified by H&E staining, were positive for von Willebrand factor. Hepatocytes were immunohistochemically positive for albumin and CK8-18-19. These results suggest that our “bio-engineered-reactor” is characterized by a steady neovasculature potentiality and keeps capacity for supporting hepatocyte viability. CONCLUSIONS: A preliminary multistep approach to generate in vivo morphologically and functionally complex new tissue has being constituted from simple monolayer. This ongoing work represents a preliminary step toward the final engineer of liver-organoid vascularized construct.INTRODUZIONE: Il progetto oggetto di questa tesi si è proposto di applicare le tecniche di ingegneria tissutale come approccio alla realizzazione di costrutti epatici per il supporto metabolico delle malattie del fegato; la realizzazione del lavoro si è sviluppata attraverso la successione multistep di diversi progetti tra loro complementari. L’ingegnerizzazione di tessuto epatico attraverso colture di epatociti primari infatti offre nuove prospettive in questo ambito; tuttavia la realizzazione di strutture 3D è spesso limitata dalla mancanza di una struttura vascolare in grado di costituire un adeguato supporto nutritivo. D’altra parte gli epatociti sono cellule ad alto metabolismo e la loro sopravvivenza è limitata in condizioni ipossiche, in mancanza di una sufficiente rete vascolare. Al fine di superare questo problema e di ottimizzare le condizioni per il supporto di nutrienti, abbiamo innanzi tutto rivolto l’attenzione alla realizzazione di un bioreattore bilayer, sviluppato nelle 2 dimensioni, e ingegnerizzato partendo da dati noti di microfluidodinamica; inoltre in grado di proporre un sistema di coltura basato sulla anatomia microscopica del fegato. Tale design ha permesso la realizzazione di un bioreattore in grado di costituire una piattaforma da utilizzare come unità di drugtesting, ma anche per la successiva realizzazione di un sistema di assistenza epatica (progetto 1). Sulla base dei dati ottenuti, il progetto è stato in seguito traslato nel modello in vivo e il bioreattore è stato testato nel piccolo animale; l’impianto del device è stato realizzato ex vivo mediante il confezionamento di uno shunt artero-venoso femoro-femorale (progetto 2). Il lavoro è stato quindi implementato sviluppando sistemi di coltura nelle 3 dimensioni (progetto 3); diverse combinazioni di coltura su scaffolds 3D in perfusione continua, all’interno di nuovi bioreattori, sono state testate per l’induzione differenziativa di cellule staminali (precursori epatocitari di origine umana). I successi ottenuti hanno spinto alla realizzazione dell’ultimo step (progetto 4). Smart scaffolds 3D di nuova generazione sono stati utilizzati per allestire colture 3D attraverso diverse combinazioni di popolazioni cellulari (epatociti primari + cellule mesenchimali staminali o soli epatociti primari), inducendo processi di neoangiogenesi. Dopo adeguato preconditioning in bioreattori in vitro, lo scaffold è stato in seguito impiantato in vivo, nel piccolo animale. METODI: Progetto 1: 18 bioreattori ingegnerizzati a flusso continuo sono stati testati per la loro capacità di trasporto di metaboliti all’interno di un compartimento parenchimale ove le colture cellulari sono state protette dagli effetti nocivi di shear stress. I bioreattori sono stati sottoposti a semina con cellule HepG2/C3A ed epatociti primari in diverse condizioni di coltura, al fine di testare la capacità del sistema di mantenere in vitro una coltura dinamica, vitale ed efficiente dal punto di vista metabolico. Progetto 2: 16 bioreattori (precedentemente ingegnerizzati nel progetto 1) sono stati utilizzati e sottoposti a coltura con epatociti primari e 12 con cellule umane HepG2/C3A; i bioreattori sono stati impiantati ex vivo in ratti atimici attraverso la realizzazione di uno shunt artero-venoso femoro-femorale. Il setting del bioreattore exvivo ha previsto diverse configurazioni, testate sotto il profilo dei parametri di vitalità cellulare sulla base dei relativi dati di pressione e di flusso nelle diverse configurazioni. Il pattern di flusso ematico e la perfusione del sistema sono stati esaminati attraverso laser Doppler scanning. La vitalità cellulare e la funzionalità metabolica sono state inoltre verificate. Progetto 3: una popolazione di Pluripotent Human Liver Stem Cells (HLSCs) è stata coltivata su 18 "smart scaffolds" 3D, composti da spugne di collagene biocompatibile. Lo scaffold è stato inserito in un nuovo bioreattore per la perfusione di strutture 3D in grado di garantire flussi uniformi di medium; medium per cellule staminali e colture miste con cellule stellate del fegato (HSCs) sono state aggiunte in diverse condizioni di coltura. Le diverse combinazioni sono state testate attraverso l’esecuzione di studi morfologici e funzionali rispettivamente nei giorni 3, 5 e 7 della perfusione in vitro. Project 4: 12 scaffolds 3D, costituiti da un derivato dell’acido ialuronico [a benzyl ester of hyaluronan (HYAFF®) sono stati sottoposti a semina con popolazioni di cellule staminali mesenchimali di origine umana (HMSCs) ed epatociti in diverse combinazioni; è stata allestita una coltura in perfusione continua nel bioreattore precedentemente descritto (preconditioning). In seguito gli scaffolds sono stati impiantati nell’omento di 12 ratti atimici, costituendo una tasca “rolled” 3D. Studi morfologici sono stati eseguiti al fine di valutare i processi di neoangiogenesis sostenuti dalle cellule HMSCs e valutare la vitalità epatocitarie a 7 giorni dall’impianto. RISULTATI: Progetto 1: Il bioreattore ingegnerizzato si è dimostrato in grado di sostenere entrambe le popolazioni cellulari in studio, comprese colture primarie di epatociti (notoriamente più sensibili), attraverso la realizzazione di una perfusione continua di medium sovrapponibile a flussi fisiologici. Tale circostanza ha favorito sia i processi di proliferazione cellulare che la funzione metabolica epatocitaria (sintesi proteica). Il modello matematico del sistema ha permesso di sostenere una coltura dinamica fino a 14 giorni. Progetto 2: Il bioreattore, collegato all’animale attraverso il confezionamento di uno shunt femorale artero-venoso, ha realizzato un sistema di perfusione ex vivo. Il flusso ematico all’interno del network vascolare si è dimostrato omogeneo nel tempo ed ha ricostituito il fisiologico pattern di flusso artero-venoso del fegato. La sopravvivenza cellulare ha dimostrato alti valori dipendentemente dai valori pressori raggiunti all’interno della camera parenchimale. La configurazione del sistema che ha generato la minore pressione all’interno della camera parenchimale ha dimostrato anche i migliori risultati in termini di sopravvivenza cellulare. Progetto 3: con l’intenzione di costituire strutture 3D, la differenziazione epatica di cellule HLSCs da fegato umano è stata indotta in scaffolds di collagene; i test morfologici e gli assays funzionali hanno confermato la maturazione. Il flusso continuo di perfusione del medium (garantito dal bioreattore) ha favorito in vitro la distribuzione delle cellule in clusters organizzati fino alle porzioni più profonde dello scaffold. Gli esperimenti hanno dimostrato che la spugna di collagene e le condizioni di coltura dinamica in vitro sono in grado di promuovere non solo la formazione di aggregati di HLSCs ma anche di favorire una più rapida maturazione verso fenotipi epatici maturi. Progetto 4: lo step finale, ancora in corso, ha previsto l’induzione della neoangiogenesi in vivo. Dopo un processo di preconditioning della coltura cellulare nel bioreattore precedentemente ingegnerizzato (progetto 3), scaffolds 3D di HYAFF® con combinazioni diverse di epatociti primari e MSCs, sono stati impiantati nei ratti. Il costrutto cosi ottenuto a 7 giorni dall’impianto ha dimostrato, alla immunofluorescenza, la formazione di iniziali clusters in cui si sono identificati eventi neoangiogenetici con formazione di strutture simil-vascolari. Gli epatociti sono sopravissuti durante tutto il periodo di studio; positiva la ricerca, alla immunofluorescenza, per albumina, CK8-18- 19. Le strutture vascolari, identificate dapprima con colorazione EE, si sono dimostrate positive alla immunofluorescenza per il fattore di von Willebrand. I dati sono suggestivi per ulteriori studi nell’ambito dei processi di neoangiogenesi. CONCLUSIONI: Gli approcci preliminari ci hanno permesso di raggiungere e ottenere in vivo risultati promettenti nella ricostituzione di costrutti epatici caratterizzati da processi neoangiogenetici, partendo da semplici layers 2D. I risultati sono suggestivi per sviluppi futuri di ingegnerizzazione di organoidi epatici

Microsoft Dynamics 365 : Business Central

reservedNella tesi di laurea vado a discutere il lavoro da me svolto durante il periodo di tirocinio e del mio approccio a Business Central, un ERP usato da Microsoft. In particolare vado a spiegare come funziona tale software, parte della sua storia (perché in azienda ho usato anche la versione precedente Navision) e quello che ho potuto sperimentare personalmente.In this bachelor's thesis i will discuss the job that i did during my internship period with Business Central. an ERP used by Microsoft. In particular i will show how this software works, the updates history (because during the internship i used the version before this, Navision) and some example about what i did