Oxidative Stress & Liver Fibrosis

By:

Khaled Elmasry

Assistant Lecturer of Human Anatomy & EmbryologyMansoura College of Medicine, Mansoura University

Mansoura, Egypt.

5/18/2014

Mechanisms of hepatic fibrogenesis:

• Multiple etiologies of liver disease lead to liver fibrosis through integrated signaling networks that regulate the deposition of extracellular matrix.

• This cascade of responses drives the activation of hepatic stellate cells (HSCs) into a myofibroblast-like phenotype that is contractile, proliferative and fibrogenic.

• Collagen and other extracellular matrix (ECM) components are deposited as the liver generates a wound-healing response to encapsulate the injury.

• Sustained fibrogenesis leads to cirrhosis, characterized by a distortion of the liver parenchyma and vascular architecture.

• Uncovering mechanisms that underlie liver fibrogenesis forms the basis for efforts to develop targeted therapies to reverse the fibrotic response and improve the outcomes of patients with chronic liver disease.

• Liver fibrosis is a reversible wound-healing response to either acute or chronic cellular injury that reflects a balance between liver repair and scar formation.

• During acute injury, the changes in the liver architecture are transient and reversible.

• With chronic injury, there is progressive substitution of the liver parenchyma by scar tissue.

• A key discovery in understanding fibrosis has been that the hepatic stellate cell (HSC) is the primary effector cell, orchestrating the deposition of ECM in normal and fibrotic liver.

• HSC plays a pivotal role in activating the immune response through secretion of cytokines and chemokines and interacting with immune cells.

• HSCs also contribute to angiogenesis and the regulation of the oxidant stress.

Key pathways of HSC activation:

1. Growth factor signaling: PDGF signaling is among the best characterized pathways of HSC activation.

2. Fibrogenic signaling pathways:

HSCs generate fibrosis not only by increasing cell number, but also by increasing matrix production per cell.

In the normal liver, the basement membrane-like matrix of the space of Disse is comprised primarily of collagens IV and VI, which is progressively replaced by collagens I and III and cellular fibronectin during fibrogenesis.

• TGFβ1 is derived from both paracrine and autocrine sources, and is the most potent fibrogenic cytokine in the liver.

3. Chemokines, adipokines and neuroendocrine pathways.

4. Immune interactions:

• The inflammatory response plays an important role in driving fibrogenesis, since persistent inflammation almost always precedes fibrosis.

• Activated HSCs secrete inflammatory chemokines interact directly with immune cells through expression of adhesion molecules,and modulate the immune system.

5. Angiogenesis:

• Angiogenesis is a key component of the wound-healing response to hepatic fibrosis and is essential for liver regeneration.

• Conversely, it is also implicated as a promoter of liver carcinogenesis;

• therefore, a fine balance of pro- and anti-angiogenic factors maintains a healthy liver.

• Endothelial cells, in addition to contributing to angiogenesis, exert paracrine effects on HSCs through nitric oxide (NO) synthase-derived NO production.

• Activated HSCs are more sensitive to NO induced apoptosis, which may be a mechanism to counter-balance HSC expansion in injured livers.

6. NADPH oxidase/oxidant stress:

• NADPH oxidase (NOX) mediates liver injury and fibrosis through generation of oxidant stress and activation of HSCs, Kupffer cells and macrophages.

• NOX also contributes to angiotensin signaling

What Is Oxidative Stress?

• oxidative stress is well known to be involved in the pathogenesis of lifestyle-related diseases.

• Oxidative stress has been defined as harmful because oxygen free radicals attack biological molecules such as lipids, proteins, and DNA.

• However, oxidative stress also has a useful role in physiologic adaptation and in the regulation of intracellular signal transduction.

• Therefore, a more useful definition of oxidative stress may be:

“a state where oxidative forces exceed the antioxidant systems due to loss of the balance between them.”

Free Radicals, Active OxygenSpecies, and Oxidative Stress:

• Usually, an atom is composed of a central nucleus with pairs of electrons orbiting around it.

• However, some atoms and molecules have unpaired electrons and these are called free radicals.

• Free radicals are usually unstable and highly reactive because the unpaired electrons tend to form pairs with other electrons.

• The reactive oxygen metabolites thus produced are more highly reactive than the original oxygen molecule and are called active oxygen species.

• Superoxide,• hydrogen peroxide,• hydroxyl radicals, and• singlet oxygen

are active oxygen species.

Biomarkers of Oxidative Stress

• The markers found in blood, urine, and other biological fluids may provide information of diagnostic value.

• Many markers have been proposed, including:

1. lipid peroxides, and 4-hydroxynonenal as markers for oxidative damage to lipids.

2. isoprostan as a product of the free radical oxidation of arachidonic acid.

3. 8-hydroxyguanine and thymineglycol as indicators of oxidative damage to DNA.

4. hydroxyleucine, hydrovaline, and nitrotyrosine.

• Lipid peroxide was assessed in clinical samples even in relatively early studies, and the analytical methods for this substance have improved.

• Lipid peroxidation is a chain reaction by which unsaturated fatty acids (cell membrane components) are oxidized in various pathological conditions.

• When a hydrogen atom is removed from a fatty acid molecule for some reason, the free radical chain reaction proceeds.

• Among the agents that protect the body from lipid peroxidation, vitamin E is considered to be the most important.

• This vitamin has attracted attention as an antioxidant because it can scavenge lipid peroxyl radicals and hence stop the propagation of the free radical chain reaction.

• The lipid peroxyl radical removes a hydrogen atom from the phenyl group of vitamin E and the molecule that has accepted the hydrogen atom is stabilized.

• In turn, vitamin E is converted into a radical, which is also stable and less reactive.

• This antioxidant reaction protects biological membranes from injury caused by free radicals and lipid peroxides.

Oxidative Stress as a Biological Modulator and as a Signal

• Oxidative stress not only has a cytotoxic effect, but also plays an important role in the modulation of messengers that regulate essential cell membrane functions, which are vital for survival.

• It affects the intracellular redox status, leading to the activation of protein kinases, including a series of receptor and non-receptor tyrosine kinases, protein kinase C, and hence induces various cellular responses.

• These protein kinases play an important role in cellular responses such as activation, proliferation, and differentiation, as well as various other functions.

NFκB Role:

• NFκB is one of the major signal-transduction molecules activated in response to oxidant stress.

• Activation of NFκB and its target genes is crucial for induction of immune responses and cell proliferation.

• It has been reported that the NFκB pathway may regulate TGF-β1 production during the resolution of inflammation in vivo;

• Inhibition of NFκB reduced TGF-β1 expression in leukocytes has been reported.

• ROS-induced NF-kB regulates TGF-β1 expression in the HCV-infected hepatocyte.

NADPH oxidase Role:

• NADPH oxidase is a multi-protein complex producing reactive oxygen species (ROS) both in phagocytic cells, being essential in host defense, and in non-phagocytic cells, regulating intracellular signalling.

• In the liver, NADPH oxidase plays a central role in fibrogenesis.

• A functionally active form of the NADPH oxidase is expressed not only in Kupffer cells (phagocytic cell type) but also in hepatic stellate cells (HSCs) (non-phagocytic cell type), suggesting a role of the non-phagocytic NADPH oxidase in HSC activation.

• profibrogenic agonists such as Angiotensin II (Ang II) and platelet derived growth factor (PDGF), or apoptotic bodies exert their activity through NADPH oxidase-activation in HSCs.

• The demonstration that NADPH oxidase is expressed in HSC led researchers to evaluate the functional activity of this complex in liver fibrosis.

• The results demonstrate that NADPH oxidase in HSCs regulates their activation process, so that the NADPH oxidase complex regulates liver fibrogenesis in response to Ang II, PDGF or apoptotic bodies.

Role of glutathione redox status in liver injury:

• The tripeptide glutathione (GSH) is the major antioxidant and redox regulator in cells that is important in combating oxidation of cellular constituents.

• Cells spend a great deal of energy to maintain high levels of reduced GSH, which in turn helps to keep proteins in a reduced state.

• Drugs, infections, and inflammation in the liver can increase ROS generation and/or decrease GSH levels and cause a shift in the cellular redox status of hepatocytes to become more oxidized.

• An alteration of the normal redox balance can alter cell signaling pathways in hepatocytes and may thus be an important mechanism in mediating the pathogenesis of many liver diseases.

• Redox status has historically been used to describe the ratio of interconvertible reduced/oxidized forms of a molecule.

• The thiol group in the cysteine residue of GSH can become oxidized to form a disulfide (GSSG).

• Because it is difficult to measure all linked redox couples in cells, a representative redox couple is generally measured.

• GSH/GSSG is the most important and commonly measured redox couple used to obtain an estimate of cellular redox state because it is found at high levels in cells.

Regulation of hepatic GSH

1. glutamate-cysteine ligase (GCL) is the rate-limiting step in GSH synthesis that catalyzes the formation of glutamylcysteine.

2. GSH synthetase catalyzes the final step in GSH synthesis.

3. GSSG reductase utilizes the reducing power of NADPH to reduce GSSG to GSH.

4. GSH peroxidase reduces H2O2and lipid peroxides to water and alcohols using the reducing power of GSH.

5. GSH transferases detoxify xenobiotic compounds through conjugation with GSH.

MITOCHONDRIA REDOX CHANGES IN LIVER DISEASES

• Mitochondrial GSH is much more important for hepatocyte survival than cytoplasmic GSH.

• The mitochondrial redox status has profound effects on the cellular redox status as a whole, because the depletion of mitochondrial GSH can cause increased release of H2O2 from the mitochondrial matrix that can oxidize cytoplasmic proteins and affect cell signaling.

• Chronic ethanol treatment and hepatitis C infection have been shown to cause mitochondrial GSH depletion.

• Chronic ethanol treatment is believed to selectively deplete mitochondrial GSH by decreasing GSH transporter activity through ethanol-induced changes in inner membrane fluidity due to cholesterol accumulation.

Nitric Oxide in Liver Injury

• Nitric oxide (NO) is a pluripotent gaseous free radical that has been identified as an important signaling molecule in virtually every tissue in the body.

• In the liver, as in many other organs, NO has many actions and cellular sources.

• As a result, the exact role of NO in regulating cell or organ function is complex, and experimental evidence often appears to be contradictory.

• This is true for its contribution to normal physiological regulation, as well as in the development of cell or tissue injury.

• NO is either a primary mediator of liver cell injury or a potent protective mechanism against injurious stimuli.

HCV and Oxidative Stress in the Liver

Possible mechanisms by which HCV promotes deposition of collagen I and other molecules in the hepatic extracellular matrix (ECM):

1. Induction of transforming growth factor β1(TGFβ1) in

hepatocytes and Kupffer cells in ROS-dependent manner.

2. Stimulation of fibrogenesis by uptake of apoptotic bodies derived from HCV-infected hepatocytes by hepatic stellate cells (HSC).

3. third mechanism of fibrogenesis is associated with enhanced production of fibromodulin, a proteoglycan released by various types of cells including hepatocytes and HSCs.

(Fibromodulin was shown to promote proliferation, migration, and invasion of HSCs in response to oxidative stress)

Antioxidants as therapeutic agents for liver

• Despite the overwhelming evidence supporting the association of oxidative stress with liver disease, the efficacy of antioxidant therapy has been extremely difficult to demonstrate.

• Despite numerous examples of efficacy of antioxidants in animal models, currently, the best clinical evidence in support of antioxidants for liver disease is the use of vitamin E for NASH.

• There is no convincing evidence to support the use of antioxidants for Hepatitis C and there is only preliminary or equivocal evidence for alcoholic liver disease.