Current Molecular Medicine - Volume 3, Issue 6, 2003

The past century has witnessed rapid progresses in the understanding of human diseases. This has been the time when studies on the pathogenesis of diseases advance from the traditional gross anatomic-physiological approach to the molecular approach, hence the birth of the molecular medicine. The remarkable achievement in the molecular biology makes the transition possible, which has no doubt deepened our understanding and changed our approach to the treatment of the disease. This progress is particularly important for the study of liver injury. An organ with multiple complicated functions and anatomically equipped with the unique double circulation systems, liver is constantly exposed to and vulnerable to hypoxia, free radicals, toxic and infectious substances. A significant portion of liver diseases are resulted from injury from these insults. Acute injury, if not resolved properly, can evolve into long-term adverse complications that could lead to liver cancer, which has a notoriously malicious clinical course and an extremely poor survival rate. While traditional analysis of the etiology and pathogenesis of liver injury could classify it into groups of infectious, ischemic, chemical, or immunological nature, molecular analyses of the biochemical process and signaling network often identify multiple mechanisms involved in each disease identity classified by the traditional method. More importantly, similar molecular processes can be shared among different disease identities. For example, immunological destruction of viral infected-hepatocytes activates death receptorinitiated caspase cascade, mitochondria dysfunction and free radicals generation, all contributing to the final demise of the cell. However, the same molecular events are also present in liver injury caused by other etiologies, such as cholestasis, ethanol, ischemia / reperfusion or endotoxemia. Thus it becomes increasingly important to understand these molecular processes and their contribution to the initiation and evolution of the disease. It is from this point that we have edited this special issue of Current Molecular Medicine for the molecular mechanisms of liver injury. To emphasize the molecular approach for the investigation of various liver injuries, in addition to the traditional vertical way of describing selected liver injury, we have devoted significantly more pages to the common molecular mechanisms, thus providing a horizontal way to examine the molecular events in details as to how they are activated and regulated in physiological and pathological conditions. The cross point of this combined vertical and horizontal approach is naturally the focus of liver injury, i.e., the functional alternations and the demise of the hepatocyte, often in the form of apoptosis. The overview by Higuchi and Gores provides a panoramic view of the molecular mechanisms of liver injury, exemplifying this approach. With the focus on the outcome of the liver injury, we then trace the molecular mechanisms of how different stimulus leads to this process, via death receptor activation (Yin and Ding), free radicals and oxidative stress (Kessova and Cederbaum, Chen et al), mitochondria permeability transition (Kim et al), or peroxisome proliferator-stimulated metabolic alterations (Yu et al) etc. For each of them, we discuss the intracellular signaling pathways, the effector molecules and the controlling and regulatory mechanisms. These molecular events constitute the pathogenesis of the most common types of liver injury, including viral hepatitis (Nakamoto and Kaneko), inflammatory hepatitis (Tsutsui et al), ischemic / reperfusion liver injury (Kim et al), septic shock induced-liver failure (Yin and Ding, Chen et al), alcoholic liver disease (Kessova and Cederbaum) and non-alcoholic steatohepatitis (Yu et al). With the long-term adverse effects of liver injury in mind, we have also addressed the mechanism of hepatocarcinogenesis (Colemen, and Yu et al), which puts a full stop on the natural history of liver injury. By putting all these topics in a single issue of Current Molecular Medicine, we hope we can best present the impact of the progress being made in this field of live study and provide a broad and comprehensive view on the molecular mechanisms that are operated in the liver injury in an intertwined way. This may serves as a road map to a better understanding of the disease and to the development of better therapeutic agents.

Liver cirrhosis, an end-result of a wide variety of the liver diseases, is a world wide health problem. Because of its unique organ system, i.e., portal blood supply, bile formation and enterohepatic circulation, drug metabolism system, and sinusoidal lining cells such as Kupffer, endothelial and stellate cells, the liver is a target of a variety of hepatotoxic insults. Current data suggest that hepatocyte apoptosis is an essential feature contributing to liver injury in a wide range of acute and chronic liver diseases. With an improved understanding of the pathophysiological role of apoptosis in liver diseases, we are now entering an era where regulation of liver cell apoptosis is becoming a therapeutic possibility. Inhibition of hepatocyte apoptosis using a variety of different strategies may be therapeutically beneficial in liver injuries, such as alcoholic hepatitis, non-alcoholic steatohepatitis (NASH), viral hepatitis, and cholestatic liver diseases. Considering the link between hepatocyte apoptosis and liver fibrosis, inhibition of hepatocyte apoptosis may also be an anti-fibrotic therapeutic strategy. Moreover, selective induction of apoptosis of activated stellate cells would be a unique approach to induce the resolution the phase of liver fibrosis. These concepts merit further clinical and basic investigation.

The TNFα receptor super-family consists of several members sharing a sequence homology in a unique function domain, the death domain, which is located in the intracellular portion of the receptor. These so-called death receptors, including Fas, TNF-R1 and TRAIL-R1 / TRAIL-R2, are expressed on hepatocytes. When stimulated by their ligands, FasL, TNFα or TRAIL, respectively, the death receptors can activate multiple death domain-initiated apoptosis programs, including both extrinsic and intrinsic pathways. A cascade of caspases is activated, which cleave proteins important for the cell structure and function. Activation of the intrinsic pathway also leads to mitochondrial release of several apoptotic proteins and mitochondrial dysfunction, which kill the cell through both caspase-dependent and caspase-independent mechanisms. Death receptor-induced hepatocyte apoptosis contributes to the development of a number of liver diseases, including viral hepatitis, inflammatory hepatitis, Wilson's disease, alcoholic liver disease, endotoxiemia-induced liver failure and ischemia / reperfusion-induced liver damage. This article comprehensively reviews the mechanisms of induction and regulation of death receptor-initiated apoptosis in hepatocytes, examines how these molecular events affect our understanding of the pathogenesis of these diseases and further discusses the potential therapeutic application of the knowledge. We hope we can provide a cohesive and integrated perspective on the many aspects of these complicated processes.

Ethanol-induced oxidative stress appears to play a major role in mechanisms by which ethanol causes liver injury. Many pathways have been suggested to contribute to the ability of ethanol to induce a state of oxidative stress. One central pathway appears to be the induction of the CYP2E1 form of cytochrome P450 enzymes by ethanol. CYP2E1 is of interest because of its ability to metabolize and activate many toxicological substrates, including ethanol, to more reactive, toxic products. Levels of CYP2E1 are elevated under a variety of physiological and pathophysiological conditions, and after acute and chronic alcohol treatment. CYP2E1 is also an effective generator of reactive oxygen species such as the superoxide anion radical and hydrogen peroxide, and in the presence of iron catalysts, produces powerful oxidants such as the hydroxyl radical. This Review Article summarizes some of the biochemical and toxicological properties of CYP2E1, and briefly describes the use of HepG2 cell lines developed to constitutively express the human CYP2E1 in assessing the actions of CYP2E1. Regulation of CYP2E1 is quite complex and will be briefly reviewed. Possible therapeutic implications for treatment of alcoholic liver injury by inhibition of CYP2E1 or CYP2E1-dependent oxidative stress will be discussed, followed by some future directions which may help to understand the actions of CYP2E1 and its role in alcoholic liver injury.

The complex role of nitric oxide (NO) in the liver can be explained by its patterns of regulation and unique biochemical properties. With a broad range of direct and indirect molecular targets, NO acts as an inhibitor or agonist of cell signaling events. In the liver, constitutively generated NO maintains the hepatic microcirculation and endothelial integrity, while inducible NO synthase (iNOS)-governed NO production can be either beneficial or detrimental. For instance, NO potentiates the hepatic oxidative injury in warm ischemia / reperfusion, while iNOS expression protects against hepatic apoptotic cell death seen in models of sepsis and hepatitis. Anti-apoptotic actions are either cyclic nucleotide dependent or independent, including the expression of heat shock proteins, prevention of mitochondrial dysfunction, and inhibition of caspase activity by S-nitrosation. Whether NO protects or injures is probably determined by the type of insult, the abundance of reactive oxygen species (ROS), the source and amount of NO production and the cellular redox status of liver. Through the use of pharmacological NO donors or NOS gene transfer in conjunction with genetically altered knockout animals, the physiological and pathophysiological roles of NO in liver function can be explored in more detail. The purpose of this paper is to review the current understanding of the role of NO in liver injury.

Reperfusion of ATP-depleted tissues after warm or cold ischemia causes pH-dependent necrotic and apoptotic cell death. In hepatocytes and other cell types as well, the mechanism underlying this reperfusion-induced cell death involves onset of the mitochondrial permeability transition (MPT). Opening of permeability transition (PT) pores in the mitochondrial inner membrane initiates the MPT, an event blocked by cyclosporin A (CsA) and pH less than 7.4. Thus, both acidotic pH and CsA prevent MPT-dependent reperfusion injury. Glycine also blocks reperfusion-induced necrosis but acts downstream of PT pore opening by stabilizing the plasma membrane. After the MPT, ATP availability from glycolysis or other source determines whether cell injury after reperfusion progresses to ATP depletion-dependent necrosis or ATP-requiring apoptosis. Thus, apoptosis and necrosis after reperfusion share a common pathway, the MPT. Cell injury progressing to either necrosis or apoptosis by shared pathways can be more aptly termed necrapoptosis.

Among seven human hepatitis viruses (A to E, G and TT virus), hepatitis B (HBV) and C (HCV) viruses are able to persist in the host for years and principally contribute to the establishment of chronic hepatitis. During the course of persistent infection, continuous intrahepatic inflammation maintains a cycle of liver cell destruction and regeneration that often terminates in hepatocellular carcinoma (HCC). While the expression and retention of viral proteins in hepatocytes may influence the severity and progression of liver disease, the mechanisms of liver injury in viral hepatistis are defined to be due not to the direct cytopathic effects of viruses, but to the host immune response to viral proteins expressed by infected hepatocytes. In the process of liver injury, hepatocellular death (apoptosis) induced by the proapoptotic molecules of T cells activated following antigen recognition triggers a cascade of antigen nonspecific effector systems and causes necroinflammatory disease. Accordingly, the regulation of the immune response, e.g., via the cell death pathways, in chronically infected patients should prevent the development of HCC.

IL-18 is a pleiotropic cytokine and is produced by various types of cells including activated macrophages, particularly Kupffer cells. IL-18 has potential to activate inflammatory responses through induction of IFN-γ production in collaboration with IL-12. Somewhat paradoxically, IL-18 also has the capacity to induce allergic responses via induction of IL-4 production by T helper cells and to activate mast cells and basophils to release atopic effector molecules such as histamine. Indeed, IL-18 is involved in inflammatory tissue injuries, such as Crohn's disease and atherosclerosis, and also in hyper IgE and atopic dermatitis. IL-18 is particularly important for induction of experimental liver diseases. Endotoxin-induced liver injury or Fas ligand-induced hepatitis is caused by endogenous IL-18 in mice. Moreover, patients with liver diseases such as fulminant hepatitis, liver cirrhosis due to hepatitis virus infection and primary biliary cirrhosis show elevation of serum levels of IL-18, that correlates with the corresponding disease severity. Therefore, endogenous IL-18 plays a major role in induction of some types of liver injuries in mice and human. NKT cells that express both T cell receptor and NK cell marker are abundant in the liver of mice and human. Recent studies have revealed that NKT cells participate in some types of liver injuries, such as concanavalin A-induced T cell-mediated hepatitis and malaria hepatitis. In this review article, we focus on IL-18-involving liver damages and NKT-cell-mediated liver injuries.

Fatty acids are metabolized in the liver by β-oxidation in mitochondria and peroxisomes and by ω-oxidation in microsomes. Peroxisomal β-oxidation is responsible for the metabolism of very long chain fatty acids and mitochondrial β-oxidation is responsible for the oxidation of short, medium and long chain fatty acids. Very long chain fatty acids are also metabolized by the cytochrome P450 CYP4A ω-oxidation system to dicarboxylic acids. Both peroxisomal ω-oxidation and microsomal β- oxidation lead to the generation of H2O2. The genes encoding peroxisomal, microsomal and some mitochondrial fatty acid metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα). Sustained activation of PPARα by peroxisome proliferators has been shown to induce hepatocellular carcinomas in rats and mice. The peroxisome proliferator-induced carcinogenic effect has been attributed to transcriptional activation of PPARα regulated genes and the resulting excessive generation of H2O2. Evidence from mice lacking fatty acyl-CoA oxidase (AOX), PPARα and PPARα / AOX has confirmed the role of PPARα in the development of hepatocellular carcinomas. In addition, mice lacking AOX developed steatohepatitis and provided clues regarding the molecular mechanism responsible for steatosis and steatohepatitis and the role of unmetabolized AOX substrates in the activation of PPARα.

The major risk factors and etiological agents responsible for development of hepatocellular carcinoma in humans have been identified and characterized. Among these are chronic infection with hepatitis B virus or hepatitis C virus, exposure to aflatoxin B1, and cirrhosis of any etiology (including alcoholic cirrhosis and cirrhosis associated with genetic liver diseases). Both chronic hepatitis and cirrhosis represent major preneoplastic conditions of the liver as the majority of hepatocellular carcinomas arise in these pathological settings. Hepatocarcinogenesis represents a linear and progressive process in which successively more aberrant monoclonal populations of hepatocytes evolve. Regenerative hepatocytes in focal lesions in the inflamed liver (chronic hepatitis or cirrhosis) give rise to hyperplastic hepatocyte nodules, and these progress to dysplastic nodules, which are thought to be the direct precursor of hepatocellular carcinoma. In most cases, the neoplastic transformation of hepatocytes results from accumulation of genetic damage during the repetitive cellular proliferation that occurs in the injured liver in response to paracrine growth factor and cytokine stimulation. Hepatocellular carcinomas exhibit numerous genetic abnormalities (including chromosomal deletions, rearrangements, aneuploidy, gene amplifications, and mutations), as well as epigenetic alterations (including modulation of DNA methylation). These genetic and epigenetic alterations combine to activate positive mediators of cellular proliferation (including cellular proto-oncogenes and their mitogenic signaling pathways) and inactivate negative mediators of cellular proliferation (including tumor suppressor genes), resulting in cells with autonomous growth potential. However, hepatocellular carcinomas exhibit a high degree of genetic heterogeneity, suggesting that multiple molecular pathways may be involved in the genesis of subsets of hepatocellular neoplasms. Continued investigation of the mechanisms of hepatocarcinogenesis will refine our current understanding of the molecular and cellular basis for neoplastic transformation in liver, enabling the development of effective strategies for prevention and / or more effective treatment of hepatocellular carcinoma.