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We evaluated the IGF1 system in cholangiocytes of primay biliary cirrhosis (PBC) patients and investigated the relationships with apoptosis. Biopsies of PBC patients (n=32) and normal subjects (n=5) were investigated by immunohistochemistry for expression in cholangiocytes of IGF1, IGF1-R, pAKT, terminal deoxynucleotide transferase end labeling (TUNEL), Bax (proapoptotic protein), and Bcl2 (antiapoptotic protein). Whereas normal cholangiocytes were almost negative, cholangiocytes of PBC patients showed strong IHC staining for IGF1, IGF1-R, and pAKT, which increases from stage I to stage IV, where >70% of cholangiocytes were positive. Bax/Bcl2 ratio reached the highest value (4.6) in PBC stage III when apoptosis is maximal (24% TUNEL positivity), whereas it declines in stage IV (1.4) when only 7.8% cholangiocytes were TUNEL positive. In PBC stages III and IV, expression of IGF1, IGF1-R, and pAKT in cholangiocytes was directly correlated with the antiapoptotic Bcl2 and inversely correlated with proapoptotic Bax, Bax/Bcl2 ratio, and TUNEL positivity. In conclusion, cholangiocytes of PBC patients showed a marked increase in IGF1, IGF1-R, and pAKT expression involving most cholangiocytes surviving in the terminal ductopenic stage. This was associated and correlated with a balance of pro- and antiapoptotic proteins favoring survival rather than apoptosis, suggesting a major role of IGF1 system in promoting cholangiocyte survival.

As previously showed for the classical self renewing tissues (i.e., bone marrow, gut and skin), rapidly changing concepts about tissue stem cell biology identify the liver as a maturational lineage system capable to generate mature cells (hepatocytes and cholangiocytes) from a resident stem cells compartment localized near the so called Canals of Hering. At present liver transplantation is the only available therapy for end stage liver diseases and there is an ever increasing shortage of donor livers. Thus in the last decades, HPC has becoming the object of many researchers since HPC might offer a new therapeutic approach for controlling the evolution of chronic liver diseases. The aim of the present review is to update readers with the evolving concepts about hepatic stem cells biology, their characterization and isolation methods and finally their therapeutic potential.

The microvascular supply of the biliary tree, the peribiliary plexus (PBP), stems from the hepatic artery branches and flows into the hepatic sinusoids. A detailed three-dimensional study of the PBP has been performed by using the Scanning Electron Microscopy vascular corrosion casts (SEMvcc) technique. Considering that the PBP plays a fundamental role in supporting the secretory and absorptive functions of the biliary epithelium, their organization in either normalcy and pathology is explored. The normal liver shows the PBP arranged around extra- and intrahepatic biliary tree. In the small portal tract PBP was characterized by a single layer of capillaries which progressively continued with the extrahepatic PBP where it showed a more complex vascular network. After common duct ligation (BDL), progressive modifications of bile duct and PBP proliferation are observed. The PBP presents a three-dimensional network arranged around many bile ducts and appears as bundles of vessels, composed by capillaries of homogeneous diameter with a typical round mesh structure. The PBP network is easily distinguishable from the sinusoidal network which appears normal. Considering the enormous extension of the PBP during BDL, the possible role played by the Vascular Endothelial Growth Factor (VEGF) is evaluated. VEGF-A, VEGF-C and their related receptors appeared highly immunopositive in proliferating cholangiocytes of BDL rats. The administration of anti-VEGF-A or anti-VEGF-C antibodies to BDL rats as well as hepatic artery ligation induced a reduced bile duct mass. The administration of rVEGF-A to BDL hepatic artery ligated rats prevented the decrease of cholangiocyte proliferation and VEGF-A expression as compared to BDL control rats. These data suggest the role of arterial blood supply of the biliary tree in conditions of cholangiocyte proliferation, such as it occurs during chronic cholestasis. On the other hand, the role played by VEGF as a tool of cross-talk between cholangiocytes and PBP endothelial cells suggests that manipulation of VEGF release and function could represent a therapeutic strategy for human pathological conditions characterized by damage of hepatic artery or the biliary tree.

The activation of hepatic stellate cells is the conversion of quiescent cells into proliferative, contractile, and fibrogenic myofibroblasts. The alpha-smooth muscle actin is a well known marker of hepatic stellate cells activation. On the other hand, the Glial Fibrillary Acidic Protein expression is a key step in the astrocytes differentiation and constitutes the hallmark response of astrocytes to injury. Glial Fibrillary Acidic Protein expression was reported in quiescent stellate cells in vivo, with an increased expression in the acute response to injury in the rat, and a down-regulation in the chronic one. This study aims to evaluate the morphologic characteristics, distribution and percentage of Glial Fibrillary Acidic Protein expressing cells in normal and injured human livers. Human liver biopsies (n=32) were divided in three groups: 1) donors liver; 2) chronic hepatitis C-virus related; 3) post-viral hepatitis cirrhosis. Samples were immunostained with anti- alpha-smooth muscle actin and anti- Glial Fibrillary Acidic Protein antibody. Subsequently they were semi-quantitatively evaluated. Liver fibrosis was quantified by morphometric analysis. In chronic hepatitis livers and in the cirrhotic ones, hepatic stellate cells became larger with long cytoplasmic processes. The alpha-smooth muscle actin -positive stellate cells increase and expand from areas of piecemeal necrosis to the entire residual parenchyma with the progression of the fibrosis. By contrast Glial Fibrillary Acidic Protein-positive stellate cells are more evenly distributed in the earlier stages and confined to the periphery of the hepatic lobule in the advanced ones. Also some endothelial cells were Glial Fibrillary Acidic Protein positive in vessels of the expanding septa and at the edges of cirrhotic nodules. Glial Fibrillary Acidic Protein expression in the liver is probably linked to the fine modulation of the cytoskeleton during the formation of cytoplasmic processes in both stellate cells and endothelial cells. In the latter Glial Fibrillary Acidic Protein expression might be related to the remodelling of the vascular bed that occurs during the response to injuries. Otherwise in hepatic stellate cells it seems to be a marker related with the acquisition of contractile properties in a subpopulation more closely associated with precocious stages of the fibrosis.

Hepatic stellate cells have been considered the most important cell-type involved in hepatic fibrogenesis. Proliferation and differentiation of hepatic stellate cells into myofibroblast-like cells has been related to the development of liver fibrosis. The alpha-actin expressed by hepatic stellate cells was considered a marker of their activation to myofibroblast-like cell. The aim of this study is to evaluate the changes in morphology, distribution, percentage and alpha-smooth muscle actin expression of hepatic stellate cells in normal and cirrhotic livers, and to correlate activated hepatic stellate cells with the progression of fibrosis. Human liver biopsies (n=121) were divided in five groups: 1) normal livers (controls); 2) cirrhosis post-HCV hepatitis; 3) cirrhosis post-HBV hepatitis; 4) non viral related cirrhosis; 5) recurrent HCV hepatitis after orthotopic liver transplantation. Samples immunostained with anti alpha-smooth muscle actin antibody by immunoperoxidase method were semi-quantitatively evaluated. Liver fibrosis was quantified by computer image analysis on specimens stained with Masson's trichrome. In normal adult livers stellate cells were very rarely stained for alpha-smooth muscle actin. In cirrhotic livers, a strongly enhanced percentage of stellate cells expressing alpha-smooth muscle actin was detected in cirrhotic fragments with respect to the control group, with a significant correlation between alpha-smooth muscle actin positive stellate cells and the volume fraction of fibrosis. Moreover, liver biopsies of recurrent hepatitis revealed an increased number of activated stellate cells compared to normal livers, and intermediate volume fraction of fibrosis. These results confirmed that a direct correlation existed between activated stellate cells and the progression of fibrosis. Alpha-smooth muscle actin confirmed to be a reliable marker of hepatic stellate cells activation also in precocious stages of the disease.