SERPIN-B3 can contribute to fibrogenic progression of chronic liver diseases towards cirrhosis and HCC development

Background/Aims. SERPINB3 (S-B3) is a serine protease inhibitor up-regulated in cirrhosis and in a significant percentage of cirrhotic patients carrying hepatocellular carcinoma (HCC). Previous studies have outlined a significant correlation between TGF-β1 and S-B3 in liver biopsies from chronic hepatitis or cirrhotic patients, with both proteins correlating to the extent of liver fibrosis. Moreover, S-B3 can stimulate epithelial-to-mesenchymal transition (EMT) and increased invasiveness of human liver cancer cell lines, two critical features that are also elicited by hypoxia, which is very common during fibrogenic progression of chronic liver diseases (CLD) towards cirrhosis and HCC development. In the present study we have investigated i) the putative pro-fibrogenic role of S-B3 and ii) whether S-B3 expression is modulated by hypoxia related mechanisms in the overall scenario of CLD progression and HCC development. Methods. Morphological analysis as well as molecular and cell biology techniques have been employed in order to investigate the two major aims of the study in: i) liver specimens from patients with HCV-related CLD, carrying or not HCC; ii) S-B3 transgenic mice (overexpressing S-B3 in the liver) submitted to chronic CCl4 administration; iii) cultures of human activated hepatic stellate cells (HSC/MFs) or human monocyte/macrophage THP1 cell line, which were also exposed to recombinant S-B3; iv) HepG2 and Huh7 human HCC cell lines. Results. Aim 1. S-B3 expression has been detected in approx. 40-50 % of chronic HCV patients analysed, with an expression level that was found to increase progressively from F1-F2 to F4 patients (METAVIR score). In S-B3 positive specimens, S-B3 was mainly expressed in hepatocytes close to fibrotic septa and αSMA positive cells. S-B3 transgenic mice exposed chronically to CCl4 (4-6 weeks), were characterized by a significant increase in extracellular matrix deposition, αSMA-positive MFs as well as in collagen type 1 and TGF-β1 transcripts vs wild type animals. HSC/MFs up-regulated S-B3 expression and release in response to both hypoxia and TGF-β1. Exposure of HSC/MFs or THP1 macrophages to recombinant S-B3 resulted in a significant increase in TGF-β1 expression and other fibrogenic or angiogenic mediators. Aim 2. Experiments performed in HepG2 and Huh7 cells exposed to hypoxia revealed that S-B3 expression and release is specifically up-regulated in a HIF-2alpha-dependent manner, with HIF-2alpha (not HIF-1alpha) found to efficiently binds to S-B3 promoter under hypoxic conditions (ChIP assay). Finally, immunohistochemistry and transcript analysis performed on specimens from HCV-cirrhotic patients carrying HCC outlined a significant correlation between HIF-2 and S-B3 expression in these human settings. Conclusions. S-B3 is likely to have a significant role in fibrogenic progression of CLD and its expression is up-regulated by either TGF-β1 as well as by hypoxia through the involvement of HIF-2alpha - related mechanisms.