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Scarpa, Melania (2008) Ruolo del fattore di trascrizione Snail1
nel processo d'attivazione delle cellule stellate epatiche.
[Ph.D. thesis]

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Abstract (english)

Liver fibrosis, the final consequence of several types of chronic hepatic injuries, results from chronic damage to the liver in conjunction with the accumulation of fibrillar collagens and ECM proteins, that alters the hepatic architecture. Hepatic stellate cells (HSC) play a pivotal role in the fibrogenic process. These perisinusoidal cells normally contribute to the maintenance of liver homeostasis by secreting growth factors, storing Vitamin A and regulating ECM turnover, and therefore play a key role to maintain the integrity of the space of Disse. Following liver injury, HSC undergo a process of activation, characterized by
morphological and functional changes and acquire a myofibroblast-like phenotype. Activated HSC proliferate and alter ECM composition both qualitatively and quantitatively. The
individuation of factors responsible for HSC transdifferentiation is therefore a fundamental step to understand the physiopathology of hepatic fibrosis.
Snail1 belongs to the Snail gene family of transcription factors, best known for its ability to trigger ephitelial-mesenchymal transition (EMT), converting epithelial cells into mesenchymal cells with migratory properties. Snail genes influence both tissue formation during embryonic development and the acquisition of invasive properties in epithelial tumors.
Snail1 is also a potent survival factor. A number of studies have reported Snail1 overexpression in several pathological conditions associated with the deposition of fibrotic tissue induced by TGF-beta, a potent Snail1 activator and profibrogenic cytokine for HSC. We therefore started examining Snail1 involvement in the hepatic fibrosis process.
To verify if HSC in vitro activation was associated to an increase of Snail1 expression, HSC purified from mice livers have been seeded on uncoated plastic and maintained in culture up to 10 days; mRNA levels of Snail1 and of the activation marker genes alpha-SMA and Col1alpha1 have been assessed by quantitative Real Time RT-PCR. This analysis evidenced that Snail1 expression augments proportionally to the activation state of HSC. Furthermore, we analysed Snail1 localization in HSC by immunocytochemistry, examining cells in culture up to 10 days: we could detect Snail1 nuclear translocation in cells in culture from at least 4 days. Next, we characterized Snail1 expression in vivo using CCl4 treatment as chronic liver injury model. Fibrosis progression has been evaluated by histology and quantitative Real Time RT-PCR
for alpha-SMA and Col1alpha1. Fibrosis development was associated to a significant increase of Snail1 mRNA. Double staining for Snail1 and alpha-SMA in fibrotic livers showed their colocalization in fibrotic septa, suggesting that activated HSC express Snail1 in vivo. Moreover, in in vivo activated HSC Snail1 resulted significantly up-regulated compared to quiescent
HSC, and immunocytochemistry analysis revealed a nuclear localization. Thus, we started functional studies by RNAi and we optimized Snail1 knockdown in primary cultures of HSC
with adenoviral vectors coding shRNA. The data obtained support the role of Snail1 in HSC activation process, since its silencing results in a decrease of mRNA levels of the activation
markers alpha-SMA and Col1alpha1 and of genes involved in the activation process such as ILK and MMP-9. Preliminary data highlight an increase of Snail1 specific transcripts in human fibrotic liver samples compared to healthy controls.
All in all, our data support a role for Snail1 in the development of hepatic fibrosis and evidence its involvement in HSC activation process.

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EPrint type:Ph.D. thesis
Tutor:Martines, Diego
Supervisor:Castagliuolo, Ignazio
Data di deposito della tesi:January 2008
Anno di Pubblicazione:January 2008
Key Words:fibrosi fegato cellule stellate epatiche snail1 attivazione
Settori scientifico-disciplinari MIUR:Area 06 - Scienze mediche > MED/12 Gastroenterologia
Struttura di riferimento:Dipartimenti > pre 2012 - Dipartimento di Scienze Chirurgiche Gastroenterologiche "Pier Giuseppe Cevese"
Codice ID:457
Depositato il:25 Sep 2008
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