Morphological characterization of liver fibrogenesis in animal models with genetically modulated TGF-beta signal transduction

Production of fibrous scar tissue in the liver is a normal response to injuries. In fibrosis this healing process is disturbed. Liver fibrosis is defined as the accumulation this fibrous scar tissue in the liver. After hepatocyte damage due to e.g. viral infection, heavy alcohol consumption, toxins or trauma, the immune system is activated and the cell repair process of the liver is induced. Injury or death (necrosis) of hepatocytes further stimulates inflammatory immune cells to release cytokines, growth factors, like TGF-beta activating hepatic stellate cells (HSCs) to produce collagen, glycoproteins (such as fibronectin) and proteoglycans. Activatied HSCs differentiate into myofibroblasts expressing alpha-smooth muscle actin (alpha SMA) which are deposited in the liver to form the extracellular matrix (ECM). At the same time, the process of breaking down or degrading collagen is impaired. In a healthy liver, synthesis (fibrogenesis) and degradation (fibrolysis) of matrix tissue are in balance. Fibrosis occurs when scar tissue is generated in excess and faster than it is degraded and removed from the liver. TGF-beta is known to play a central role in regulation of the production, degradation, and accumulation of ECM proteins and that, as a consequence, it may play a pivotal role in the fibroproliferative changes that follow tissue damage in many vital organs, including liver, lung, kidney, skin, heart, and arterial wall.(Massague, J. 1990). Therefore numerous antifibrotic strategies aim to block action of this cytokine or reduce its synthesis. TGF-beta superfamily members bind to serine/threonine kinase receptors. expressing on surface of a variety of cells e.g. hepatocytes and HSCs. TGF-beta receptors comprise two structurally similar subfamilies, the type I and type II receptors, both with small cysteine rich extracellular regions and intracellular portions consisting mainly of their kinase domains. To transmit a signal, type II and type I receptors act in sequence (Wrana et al., 1994). TGF-beta first binds to T beta RII, which resides in the cell membrane in an oligomeric form with intrinsic kinase activity; T beta RI is then recruited and phosphorylated in its GS domain by T beta RII, leading to activation of its kinase activity and subsequent intracellular signaling (Massague 1998 and Piek et al.,1999). Smad7 belongs to inhibitory Smad propteins.(I-Smad) exerting their inhibitory effect on TGF-beta by binding to the T beta RI to hinder posphorylation of Smad2 (receptor Smad, R-Smad) and subsequent signaltransduction pathway to nucleus. Because the expression of Smad7 is upregulated by TGF-beta, this molecule is an important feedback inhibitor of TGF-beta signaling (Dooley et al., 2003). Since Smad7 represents an efficient inhibitor of TGF-beta signaling, it is conceivable that hyperexpression of Smad7 is an additional means to antagonize TGF-beta in fibrogenesis. In a part of my own work, we studied the effect of Smad7 overexpression after adenoviral gene transfer (AdSmad7) in primary cultured HSCs and in vivo after BDL of rats (Dooley et al., 2003). Exogenous Smad7 overexpression prevented BDL dependent activation of HSCs and liver fibrosis. Collagen I deposition and hydroxyproline content were significantly reduced in Smad7 overexpressing livers.. As AdSmad7 viruses infected and resided in all cell types of the liver, specificity of its effect could not be related to a single cell type. Even an influence on other organs could not be excluded. In my thesis I therefore established an animal model with inducible overexpression of transgenic Smad7 in hepatocytes (S7tg mice) and studied its effect on fibrogenesis after CCl4 induced liver damage. In contrast to albumin/TGF-beta 1 transgenic mice of Sanderson and colleagues (Sanderson et al., 1995) the mortality rate of S7tg mice was infinitely small during the CCl4 treatment protocol I used. In S7tg mice the Smad7 gene was expressed under the control of the human CRP promoter. CRP is a component of the congenital immune system stimulated by LPS. Binding of LPS to cell surface receptors triggers the synthesis of inflammatory cytokines including IL-6, which then induces expression of CRP (Agrawal et al., 2001). Because CRP and its transcription factors are mainly expressed in hepatocytes, this new mouse model leads to specific Smad7 expression in hepatocytes. These mice and corresponding controls were treated with CCl4 for 8 weeks. In addition, mice with a disrupted Smad7 gene (S7 Delta E1) were investigated. The S7 Delta E1 strain displayed the highest degree of fibrosis, indicating that the presence of a functional negative feedback regulation for TGF-beta signaling is controling the degree of fibrogenesis. Interestingly, Smad7 overexpression in hepatocytes of CRP-Smad7 transgenic mice was sufficient to reduce fibrosis and expression of protein markers in the liver, e.g., collagen and alpha-SMA, as well as in serum, e.g., aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (AP) and hyaluronic acid (HA), by more than 70% in comparison to wild type animals. Further, Smad7 overexpression reduces proliferation, inflammation and apoptotic processes in liver cells of CRP-Smad7 transgenic mice. Taken together, my thesis demonstrates that TGF-beta signaling in hepatocytes is required for progression of chronic liver disease and blocking its signaling pathway in this cell type by ectopic expression of Smad7 is sufficient to obtain a beneficial outcome