Working mechanism of Hic-5 in human and experimental NAFLD-related liver fibrosis

Non-alcoholic fatty liver disease (NAFLD) is characterized by a wide spectrum of pathological findings, ranging from simple steatosis to steatohepatitis which can progress to cirrhosis and hepatocellular carcinoma. Fibrosis severity is the main determinant of liver-related complications and mortality, therefore its regression is considered an important therapeutic endpoint in clinical trials evaluating pharmacological intervention in NAFLD. Activation of hepatic stellate cells (HSC) by transforming growth factor beta (TGFβ) is a well-established driver of liver fibrogenesis. Recently, a TGFβ-modulated protein, namely hydrogen peroxide-inducible clone 5 (Hic-5) has been proposed as a novel potential therapeutic target for liver fibrosis, since its knockdown attenuated experimental liver fibrosis in mice. However, limited data are available regarding its expression in human HSC, while no study has investigated its role in the setting of NAFLD so far. Based on these premises, the aim of my thesis was first to characterize Hic-5 expression in human HSC according to different hepatic microenvironment (healthy versus fibrotic); secondly to assess whether Hic-5 is expressed in human and experimental NAFLD. To investigate the first aim, primary human HSC were cultured in a new 3-dimensional (3D) culture model based on decellularized human liver extracellular matrix (ECM) scaffolds derived from healthy and cirrhotic livers. Compared to traditional 2D cultures, Hic-5 gene expression was significantly upregulated in HSC cultured in the 3D model. This effect was further enhanced upon TGFβ stimulation in healthy scaffolds. Moreover, higher Hic-5 mRNA levels were detected in HSC cultured in cirrhotic scaffolds compared to healthy scaffolds, suggesting that Hic-5 expression and its modulation by TGFβ are strongly affected by liver- and disease specific ECM features. Based on these preliminary in-vitro results, I decided to further investigate Hic-5 expression in human NAFLD samples obtained from obese patients undergoing bariatric surgery. Immunohistochemical staining showed an increased expression of Hic-5 in fibrotic liver tissue, which overlapped alpha smooth muscle actin (α-SMA) positive areas. In order to better elucidate the role of Hic-5 across NAFLD progression, a nutritional rat model of NAFLD, based on the administration of choline-deficient (CD) diet, was employed and hepatic Hic-5 expression analyzed at different time points. After 3 days of CD diet Hic-5 mRNA levels were not significantly upregulated, suggesting that fatty liver alone without associated liver fibrosis does not affect Hic-5 expression. Accordingly, no difference in α-SMA gene expression was observed compared to control diet. In contrast, administration of CD diet for 7 weeks led to increased mRNA expression of Hic-5 along with αSMA, TGFβ1 and collagen type 1A1. Upregulation of Hic-5 was further confirmed at the protein level by Western blot analysis. Similar findings were obtained when CD diet was given for 11 weeks. Interestingly, the administration of a triiodothyronine(T3)-supplemented diet for 1 week, which has been previously shown to revert rat fatty liver, was able to reduce the expression of Hic-5. The same effect of T3 on Hic-5 was seen on the microenvironment surrounding preneoplastic nodules during NASH-related experimental liver carcinogenesis. Since T3 administration was associated with regression of preneoplastic nodules not expressing Hic-5 or αSMA, we speculate that its antifibrotic effect may contribute to preneoplastic nodules regression. Taken together, these results highlight the role of Hic-5 in HSC activation in vitro and its association with NAFLD progression. Further studies are needed to clarify the molecular mechanisms regulating Hic-5 expression in NAFLD-related fibrosis and the effects of thyroid receptor’s agonists in order to identify potential anti-fibrotic therapie