In this connection, these authors showed that the process of HSC activation is accompanied by remarkable changes blog post in the expression of some key proteins involved in the control of apoptosis, and in particular, a shift towards a higher Bcl2/ Bax ratio protein expression. Based on this initial report, the aim of the present study was to further characterise the pathways modulating the apoptotic process in activated human HSCs. In order to maximise this effort, the expression and regulation of dif ferent cytoplasmic and nuclear protein systems were eval uated before and following stimulation with IGF I, a factor known to support growth, metabolism, differentia tion and prevention of apoptosis in many cell types. Although IGF I is produced by many tissues, liver IGF I synthesis accounts for 90% of the circulating peptide.
In particular, liver IGF I is synthesised at high levels in hepa tocytes in response to growth hormone stimulation, and in multiple non parenchymal cell types including HSC. These cells express IGF I receptor and are important targets for IGF I action. In cultured HSCs, IGF I enhances proliferation, migration and collagen synthesis, providing indirect evidence that IGF I could play a role in the expansion of activated HSCs and liver fibrosis. In previous studies, we investigated the intracellular pathway of human HSCs involved in both the mitogenic and chemotactic effects. In particular, it was shown that the activation of PI 3K and ERK is required for both IGF I dependent HSC proliferation and chemotaxis, confirm ing an interaction between PI 3K/Akt and MAPK/ERK pathways.
The aim of this study was to investigate Brefeldin_A the intracellular survival signal induced by IGF I and its pos sible biological effect. Materials and methods Materials Enhanced chemiluminescence reagents and nitro cellulose membrane Hybond C extra were from Amer sham Pharmacia Biotech,IMMOBILON Western reagents were from the Mil lipore Corporation IGF I and platelet derived growth factor from Peprotech EC Ltd, Fas ligand from Upstate Biotech. Antibody against Bad, Akt and poly polymerase were from Santa Cruz Biotechnology, all other antibodies were from Cell Signaling Tech nology. Iscoves medium was from Invitrogen. Annexin V FLUOS stain ing kit was from Roche. All other reagents were from Sigma Chemical Co.
Cell isolation and culture The use of human material was approved by the Human Research Review Committee of the University of Florence, where apply for it cells were isolated and characterised from surgical wedge sections of human livers not suitable for transplan tation, as described elsewhere. Cells obtained from samples of different normal human livers were cultured in Iscoves medium supplemented with 20% foetal bovine serum. After reaching confluence in the primary culture, serial passages were obtained, always applying a 1 3 split ratio.