This presentation introduces a brief and rapid review for an important research area (oxidative stress) and its relation to liver fibrosis. Liver fibrosis is very important for us as we are facing a very dangerous and continuously growing problem in Egypt, HEPATIC PATIENTS COMPLICATIONS.

1. Oxidative Stress & Liver Fibrosis
By:
Khaled Elmasry
Assistant Lecturer of Human Anatomy & Embryology
Mansoura College of Medicine, Mansoura University
Mansoura, Egypt.
5/18/2014

2. 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.

3. • 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.

4. • 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.

5. 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.

6. 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.

7. 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.

8. 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

9. 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.”

10. Free Radicals, Active Oxygen
Species, 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.

11. 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.

12. • 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.

13. 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.

14. 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.

15. 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.

16. • 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.

17. 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.

18. • 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.

19. 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.

20. 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.

21. 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.

22. 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)

23. 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.