Etiology and epidemiology of chronic liver diseases
Chronic liver diseases (CLD) and their end-stages, liver cirrhosis and hepatocellular carcinoma (HCC), are leading causes of morbidity and mortality worldwide. Patients with liver cirrhosis are at high risk of hepatic failure and more than 80% of HCC develop on a background of liver cirrhosis. HCC is the fifth most common cancer worldwide, accounting for at least 600,000 deaths annually and thus is a major global health problem. The main etiologies of CLD in industrialized countries include chronic HCV infection, alcohol abuse and non-alcoholic steatohepatitis (NASH). The complex multistep process of a progressive CLD is reflected in modulated intracellular signal transduction, changed cell communication and more drastically in an altered differentiation state of most cell types.
The liver posseses an enormous regenerative capacity. In experimental partial hepatectomy (PHx) models, rats or mice restore the mass of the liver within 5-6 days after two-thirds PHx. However, in some settings, potency of the toxic xenobiotic or the amount of cells ingested may cause death of a large number of hepatocytes and cholangiocytes. In this setting, hepatic progenitor cells (HPCs), residing in the ductules and canals of Hering, with bi-potential capacity, differentiate into either hepatocytes or cholangiocytes, depending on the cell compartment that is damaged most. If hepatic regeneration based on mature hepatocytes/cholangiocytes and HPC proliferation cannot replenish dead liver cells and liver architecture, hepatic failure soon occurs.
Hepatocellular carcinoma (HCC) formation
Recent analyses using genome-wide approaches and improved animal models have initiated new and promising attempts at subclassifying the apparently highly heterogeneous HCC into distinct molecular and prognostic subtypes. While the generally accepted paradigm of hepatocarcinogenesis is that malignant transformation occurs through the stepwise accumulation of genetic and epigenetic alterations that lead to the progressive acquisition of the cancer phenotypes, recent studies have led to the concept that a minimum number of molecular alterations leads to the acquisition of the key cancer phenotype - unconstrained cell proliferation. This “cancer platform” concept proposes that in a physiological context, growth-promoting pathways are coupled with the activation of control mechanisms such as cellular senescence or apoptosis that limit their growth effects, producing a natural homeostasis of tissue mass. Inactivation of this “oncogene-induced survey and death” during oncogenic events results in unconstrained proliferation.
Current therapy for CLD
Liver transplantation is currently the only available treatment for terminal liver failure. Since donor organs are highly limited, there is a strong interest in new therapies. Current efforts aim at discarding the source of damage, which represents the most efficient strategy of healing. In alcoholic liver disease (ALD), this can be achieved by avoiding further alcohol consume, which is complicated by addiction behaviour of the patients. Regarding HBV or HCV infections, which represent about 30% of CLDs, virostatic treatments are currently in use to decrease virus load, thus improving patient conditions to some extent. However, the fact that virus infection cannot be completely eradicated narrows efficiency of this therapy. Add on approaches that target proinflammatory and profibrotic signalling pathways activated during and required for disease progression are promising candidates to provide medical treatment.
Molecular mechanisms associated to CLD initiation and progression
Cell damage and death
CLD are characterized by persistent hepatocyte damage and death, induced by either chemical toxicity, metabolic overload resulting in high levels of reactive oxygen species (ROS), or viral/microbial activity causing metabolic de- regulation. Several modes of cell death have been classified in the damaged liver, including apoptosis and necrosis. Hepatocyte death triggers a cascade of reactions that is geared towards damage limitation, removal or repair of damaged cells, defence against further infection as well as tissue repair and regeneration.
The central process of CLD is inflammation, induced by a battery of signalling molecules and executed by a variety of cells. When chronic injuries occur, inflammation is characterized by a large infiltration of mononuclear cells, which include macrophages, lymphocytes, eosinophils and plasma cells. In these cases, lymphocytes are mobilized and stimulated by contact with antigen to produce lymphokines that activate macrophages. Cytokines and chemokines from activated macrophages, in turn, stimulate lymphocytes, thus setting the stage for persistence of an inflammatory response.
The cell responsible for cell repair, and subsequently fibrosis in any CLD appears to be the activated myofibroblast. There are several potential sources of this critical mediator, including bone marrow-derived fibrocytes or circulating mesenchymal cells, which can migrate through the injured liver and become myofibroblasts. Resident cells, e.g. tissue fibroblasts located in the portal tract of the liver or quiescent hepatic stellate cells (HSC) located in the Space of Disse, can be activated to become myofibroblasts. It is still under controversial discussion whether hepatocytes, cholangiocytes, or even endothelial cells may undergo a transition into activated myofibroblasts. The predominance of evidence supports a central role of the quiescent HSC becoming activated by cytokine signalling, turning to a myofibroblast and then producing the fibrous scar found in CLD. Based on a group of results with rat or mouse HSCs and animal models of liver damage, five critical statements about liver fibrosis can be drawn: (1) Oxidative stress induces liver fibrosis; (2) hepatocyte loss induces hepatic fibrosis; (3) TGF-β is required for liver fibrosis; (4) chronic inflammation leads to fibrosis; (5) apoptosis of HSCs prevents and reverses fibrosis.
In cirrhotic livers, macroregenerative nodules with foci of hepatocyte dysplasia have been identified as preneoplastic lesions of HCC. Histologically, dysplastic lesions are classified as small cell or large cell lesions or as foci of adenomatous hyperplasia. Available evidence suggests that small cell dysplasia and adenomatous hyperplasia are the predominant preneoplastic lesions. Our present lack of a unified, comprehensive understanding of liver carcinogenesis is partially due to the fact that HCC is initiated in multiple genetic and environmental contexts, and almost certainly emerges as a consequence of multiple pathways. This lack of understanding regarding the pathogenesis of HCC has also prevented the development of effective, targeted, preventive, or therapeutic interventions.
The Crucial Role of TGF-β
TGF-β represents a key cytokine that shows up in liver in its activated form upon damage and from then on triggers important cellular events during any progression stage of the disease. TGF-β regulates fibrogenesis and repair in any kind of CLD. Most liver cells are sensitive to TGF-β and able to trigger downstream signalling in response to this cytokine. TGF-β triggers activation of HSC, mediates ECM synthesis, provides cell contraction and migration, induces hepatocyte apoptosis and its signalling induces oxidative stress. Regulatory T cells and Th17 cells, both important negative regulators of inflammation, depend on TGF-β for terminal differentiation, indicating its impact in the inflammatory response. TGF-β induces cell death and epithelial mesenchymal transition of hepatocytes, both facilitating extracellular matrix deposition and scar formation. Cytostatic and apoptotic functions of TGF-β on hepatocytes are critical for the control of liver mass and their loss may lead to hyperproliferative disorders and cancer. Thus, TGF-β displays tumor suppressor functions in early stages of liver damage and regeneration and may turn to a tumor promoter during cancer progression. Finally, activation of liver sinusoidal endothelial cells and neoangiogenesis is partially facilitated by TGF-β. While the role of TGF-β as master cytokine controlling CLD is established, the complexity of the underlying response of the liver cells and in the liver as a whole leading to the drastic changes observed is currently not understood.
Targeting TGF- β signalling in CLD
Anti TGF-β approaches were established and successfully used for the treatment of experimental fibrogenesis. Dominant negative TGF-β receptors (TβR) were applied to suppress fibrosis. Similarly, TGF-β binding proteins like decorin, antagonistic cytokines such as bone morphogenetic protein-7, hepatocyte growth factor, IL-10, or IFN-γ were as efficient as camostat mesilate, a protease inhibitor that possibly abrogated proteolytic activation of TGF-β. Further, Dr Dooley’s group (partner of this consortium) recently overexpressed Smad7 in bile duct ligation induced liver fibrosis and achieved efficient inhibition of intracellular TGF-β signalling, thereby counteracting profibrogenic effects in cultured HSCs and in vivo. Based on the aforementioned approaches, a cohort of studies were performed aimed to abrogate fibrogenesis in different fibroproliferative diseases, including liver, and most of these were very promising. Contrary to the stimulating beneficial outcome of anti-TGF-β treatment in animal disease models, only limited positive or even adverse results exist for human disease. For example, Metelimumab, a monoclonal antibody against TGF-β1 was used to treat systemic sclerosis. The study was stopped since 4 patients died. Other studies using Lerdelimumab, a monoclonal antibody against TGF-β2, to treat eye scarring, or GC1008, a pan-antibody against TGF-β1-3 for lung fibrosis are still ongoing. Intriguingly, an antisense strategy against TGF-β2 was successful in treatment of glioma and is currently tested in other malignant tumours like pancreatic carcinoma, colon carcinoma and melanoma. A major reason why most of these promising results from animal disease models have not yet robustly been translated into clinical use, is the multiplicity of possible biological functions of cytokines. When looking at TGF-β as representative for other cytokines, complexity starts at the network of downstream signal transduction. Besides the canonical pathway, which uses TGF-β receptor II/ALK-5 dependent activation of Receptor (R)-Smads2 and 3, complex formation with Co-Smad 4 and subsequent nuclear translocation of a transcriptionally active Smad2/3/4 complex, in different cell types, including HSC and hepatocytes, TGF-β may as well signal via ALK-1 and subsequent phosphorylation of receptor Smads1, 5 and 8. Furthermore, Smad-independent signalling may occur downstream of TGF-β receptor activation and transmit the signal via MAP-kinase- (p38, ERK, JNK), PI3K- (Akt) or NF-κ-B-pathways. An additional challenge of anti-cytokine treatment for fibroproliferative diseases is to select the right time point of intervention. In the time course of CLD progression, there are different phases, such as initiation, regeneration, perpetuation, fibrogenesis, tumorigenesis and metastasis. Depending on the specific disease stage, TGF-β may have adverse or beneficial outcomes as outlined briefly below. Initially, TGF-β enhances hepatocyte damage. On the other hand, it triggers transdifferentiation of HSC to MFB and thus mediates a wound healing response. During regeneration and hepatocyte proliferation, TGF-β has an important tissue-mass limiting cytostatic effect and it controls inflammation by generating T-regs. During perpetuation and fibrogenesis in chronic disease stages, the scar forming overwhelming wound healing reaction is adverse for the liver. In the pre-malignant stage, the cytostatic effect that controls epithelial cell proliferation may prevent tumorigenesis. On the other hand, the negative outcome of TGF-β on inflammation may inhibit the immune response against arising tumour cells. In tumorigenesis, when TGF-β-cytostatic effects are lost and the signalling branch is redirected to EMT, TGF-β may favour cancer progression and metastasis. And finally, pro-angiogenic action of TGF-β towards endothelial cells may as well be important for tumour progression.
Strategies to inhibit TGF-β signalling
Various strategies have been pursued to accomplish inhibition of TGF-β signalling including (i) sequence-specific anti-sense oligonucleotides for degradation of TGF-β mRNA, (ii) isoform-selective, neutralizing antibodies or soluble TGFBRII fragments blocking the binding of TGF-β ligands to the heteromeric receptor complex, (iii) synthetic peptides that block the interaction with the membrane receptors and (iv) low molecular weight inhibitors antagonizing the intracellular kinase activity of TGFBRs. For instance, the small molecule inhibitor LY2109761 targeting TGFBRI-ALK5 and TGFBRII induced a complete abrogation of Smad-dependent and - independent signalling in human colon carcinoma cells harboring activated K-RAS, resulting in reduced tumor cell invasion and liver metastasis. Remarkably, the company DIGNA, member of this consortium, has recently developed selective inhibitors of TGF-β, blocking the interaction with the membrane receptor, using synthetic peptides. One of these peptides has proven effective on the protection against CCl4-induced liver fibrosis model. Futhermore, recent studies in Dr. Giannelli’s lab (member of this consortium) have shown that inhibition of TGF- β signaling results in multiple synergistic downstream effects which will likely improve the clinical outcome in HCC.