Liver

Review of Literature
Liver
The liver is the second largest organ in the human body after skin (Racanelli and
Rehermann 2006).and has been considered second only to brain in its complexity.
The liver has a unique role in the body, as it is the central organ for metabolism (e.g.
of lipids, carbohydrates, amino acids and lipoproteins), for biosynthesis of serum
proteins and coagulation factors and for storage of glycogen, triglycerides, and
vitamins. The liver also is the largest secretory gland of the body, producing bile that
is secreted into the bile ducts. Furthermore, through production and degradation of
hormones, the liver is part of the endocrine system . and is the major site for
biotransformation (particularly making hydrophobic molecules water soluble)
(Malarkey et al., 2005).
It is located between the portal and general circulation ,between the organ of
gastrointestinal tract and the heart . The liver is the first organ to be reached by the
blood from the intestines. It receives a dual blood supply; approximately 20% of the
blood flow is oxygen rich blood from the hepatic artery , and 80% is nutrient rich
blood from the portal vein
araising from the stomach, intestine and spleen
(Mahtabmamun and rhmansalimur 2010). Not only The portal blood is enriched
with nutrients but also contains pathogens and toxins. Therefore, the liver exerts
important functions of the innate and adaptive immune system and is responsible for
detoxification(Lalor and Adams 2002).
Functionally, the liver is organized in liver lobules, each comprising all hepatic
cell types. These are hepatocytes, which are the hepatic epithelial cells, endothelial
cells, and resident non-parenchymal cells including hepatic stellate cells (HSCs) and
Kupffer cells (KCs). Hepatocytes, representing the major cell type in the liver, are
responsible for metabolism and detoxification. They are provided with oxygen,
nutrients, toxins and the bodys own waste products by a dual blood supply system.
The hepatic artery supplies the cells with oxygenated blood whereas nutrient-rich
] 1[
blood from the intestines is delivered by the portal vein. The blood passes through the
widely branched sinusoids, which are the microvascular units of the liver. This
guarantees a maximum exchange with the parenchymal cells before the blood enters
the central vein to finally return to the circulation through the hepatic vein. Biles
produced by hepatocytes are removed through the bile ducts. Figure 1-1
schematically illustrates the architecture of a liver lobule (Lalor and Adams 2002).
The sinusoids are lined by a discontinuous layer of liver sinusoidal endothelial cells
(LSECs). These are fenestrated with pores, which are grouped in sieve plates. These
fenestrae occupy 6-8% of the surface and serve to sieve the fluids that are exchanged
be-tween the lumen of the sinusoids and the perisinusoidal space (Hernandez-Gea
and Friedman 2011;Wisse et al., 1996).The lumen of the sinusoids accommodates
Kupffer cells, which are resident macrophages, and Pit cells, which are liver-specific
natural killer cells (Wisse et al., 1996).The sinusoids lack a basement membrane
underlying the epithelial layer. This allows for effective exchange of fluid between
sinusoids and the surrounding cells. However, the space underlying the epithelial cells
contains a basement membrane-like matrix with low density. This perisinusoidal
space is called the space of Disse. The space of Disse contains HSCs, which comprise
15% of the resident cells of the healthy liver (Friedman 2000). The ratio between
HSCs and hepatocytes is about 1:20 (Moreira 2007). HSCs are named according to
their star-like appearance. In the healthy liver, they express a quiescent phenotype and
represent the bodys largest reservoir of vitamin A (about 80%), which they take up
by receptor-mediated endocytosis and store it as retinyl esters in large perinuclear
lipid droplets (Smedsrodet al., 2009).
HSCs furthermore maintain the steady-state of the membrane-like matrix in the
healthy liver while substantially contributing to excess matrix deposition in liver
fibrosis (Moreira 2007). HSCs account for 5% to 8% of total liver cells , and their
functions include vitamin A homeostasis; ECM synthesis and degradation; sinusoidal
blood flow regulation ; erythropoietin expression in the perinatal period ; contribution
] 2[
to the plasminogen activation system ; and secretion of paracrine, juxtacrine,
autocrine, and chemoattractant mediators (Su et al., 2014).
FIBROSIS wich IS A NONPHYSIOLOGICAL( Zeisberg and
Kalluri 2013)
scarring process is a common feature of most chronic diseases. In epithelial organs,

especially the lung, liver, skin, and kidney, the replacement of normal functional units
of cells with collagen-rich scar tissue and the architectural distortion caused by scar
retraction are major factors in progressive loss of organ function and eventual failure.
Because each of these organs has a different purpose, is exposed to diverse
environmental factors, and is composed of different cell types, there are unique
features and consequences of tissue fibrosis among these organs. There are also,
however, core features shared by pathologic fibrosis among multiple organs
(Friedman et al., 2013).
Fibrotic diseases, such as liver, pulmonary, and renal fibrosis, are common endstage conditions that are major health problems worldwide and put significant strain
on the health-care systems of numerous countries. (Zhou et al., 2012).
Fibrotic diseases account for up to 45% of the total deaths in developed
countries (Wynn 2008).Despite contributing to as much as 45% of deaths in the
industrialized world, fibrotic diseases have been largely overlookeduntil now
(Friedman et al., 2013).
Hepatic fibrosis (HF) is a pathological condition resulting in abnormal
proliferation and accumulation of tough fibrous connective tissue (scar tissue) in the
liver. Although the formation of scar tissue is a normal body response to injury, in
fibrosis, this healing process goes erroneous. Normal process of wound healing
involves collagen deposition; however, the chronic activation of this healing
mechanism leads to liver pathology. Among a variety of causes/factors or stimuli,
] 3[
which bring about this transformation are: chronic infection by hepatitis B, C viruses
and parasite Schistosoma, chronic alcoholism and/or exposure to certain drugs and
toxins, infections, non-alcoholic steatohepatitis (NASH), inherited metabolic diseases
like hematochromatosis, Wilson's disease, -1 antitrypsin deficiency, autoimmune
diseases such as primary biliary cirrhosis, and auto-immune hepatitis (Ahmad and
Ahmad 2012).liver fibrosis wich results from the progressive accumulation of a
qualitatively altered extracellular matrix that is highly enriched in type I and III
fibrillar collagens( Mallat and Lotersztajn 2013). which may progress to liver
cirrhosis and liver carcinoma if not reversed (Mostafa et al., 2013).
Liver fibrosis can be divided into those diseases in which the fibrosis is portal
based and those that are central based. Chronic viral hepatitis (viral and autoimmune),
chronic cholestatic diseases and hemochromatosis are the major portal based diseases
and steatohepatitis and chronic venous outflow obstruction of any cause can causes
central based diseases (Benyon and Arthur 2001).
Liver fibrosis is the final pathological outcome for the majority of chronic liver
insults. Chronic liver diseases (CLDs) are a major cause of morbidity and mortality
worldwide, affecting 360 per 100,000 persons and ranking as the 12th leading cause
of overall mortality(Sebastiani et al., 2014).Prevalence of chronic liver diseases,
hence hepatic fibrosis ,cirrhosis, is predicted to increase, due in part to the rising
prevalence of obesity and metabolic syndrome, especially in developed countries
(Duval et al., 2015).
It is extremely challenging to recognize when the liver is ill. Liver disease is not
always associated with concrete symptoms such as jaundice or acute pain,normaly the
liver is compromised for years if not decades before gradually becoming more and
more damaged with frequently fatal consequences such as liver cirrhosis and cancer.
though the causes of the CLDs are different will progress to liver fibrosis and
cirrhosis (Hernandez-Gea and Friedman 2011).
] 4[
Causes of chronic liver disease:

Although acute injury will also activate the process of fibrogenesis, liver injury
associated with chronic liver diseases is required for significant
fibrosis to
accumulate. Any chronic perturbation of hepatic homeostasis, whether visible by light

microscopy or not, may elicit the signals necessary for the initiation of fibrogenesis
(Friedman 2008). Chronic liver injury, regardless of etiology, induces liver
fibrogenesis through a dynamic and highly integrated process that leads to
progressive accumulation of extracellular matrix (ECM) components with an attempt
to limit hepatic damage. Sustained liver injury activates resident hepatic stellate cells
(HSCs), which are considered as a major source of fibrogenesis though portal
fibroblast (Beaussier et al., 2007), bone marrow derived fibrocytes (Inagaki and
Higashiyama 2012), and resident hepatocytes undergoing epithelial to mesenchymal
transition may also contribute to hepatic fibrogenesis (Zeisberg et al., 2007).
Chronic liver diseases include a number of hepatic diseases with different
pathogeny. Many conditions cause CLDs, the major reason consist virus infection,
chronic alcohol drinking, non-alcoholic fatty liver, drugs, autoimmune diseases and
genetic diseases.Though CLDs are universally reported in the world, but the
incidence of these CLDs shows some regional characteristics, In the West, the main
causes of liver fibrosis are chronic hepatitis C infection wich is the most important
liver disease in the 20th century, alcohol and NASH (non-alcoholic steatohepatitis)
wich will be the most important liver disease in the 21st century, whereas viral
hepatitis (hepatitis B and C) predominate as the major causes of liver disease in the
middle East and Asia (Henderson and Iredale 2007;Brenner 2009).
Hepatitis virus infection is one of primary threat of liver. There are five kinds of
hepatotropic viruses: Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D, and Hepatitis
E. These hepatitis viruses are unrelated; the only common ground of them is the same
] 5[
infection target, liver. The hepatitis B and C viruses (HBV and HCV) are two most
common causes of chronic viral hepatitis(Arzumanyan et al., 2013).
Chronic Hepatitis C (HCV) is one of the major causes of liver fibrosis, with
distortion of the hepatic architecture, and ultimate progression to cirrhosis.
Approximately more than 3% of the total world population is chronically infected
with HCV and due to gradual increase in the prevalence of HCV; future burden of
chronic HCV is predicted to raise at least 3 fold by the year 2020 (Ahmad et al.,
2011). HCV infection is found worldwide. Countries with high rates of chronic
infection are Egypt (22%), Pakistan (4.8%) and China (3.2%). these countries are
attributed to unsafe injections using contaminated equipment (WHO 2011).Whereas
Egypt is considered a region of intermediate prevalence (27%) of HBV infection,the
prevalence of HCV infection is the highest in the world, with 15% of the general
population having HCV antibodies (Abdelwahab et al., 2012).
Schistosomiasis is a parasitic disease caused by infection with the helminth
Schistosoma species. The arrival of eggs in the liver during Schistosoma mansoni
infection initiates a protective granulomatous response. However, as the infection
progresses, this response results in chronic liver fibrosis, a central event in
progressive liver disease. This leads to cirrhosis, and associated morbidity and
mortality caused by decompensation (Harvie et al., 2007).
Schistosomiasis is a chronic parasitic liver disease that constitutes a major
public health problem in several parts of the world. There are more than 200 million
people affected by schistosomiasis worldwide and 600 million people are at risk of
infection.Many patients exhibit high morbidity and mortality associated with
periportal fibrosis, portal hypertension and splenomegaly, which lead to frequent
episodes of upper gastrointestinal bleeding (Leite et al., 2013). In Egypt, HCV
together with schistosomal parasite infection is the biggest risk factor for chronic liver
] 6[
disease. In most Egyptian patients, HCV genotype 4 is highly prevalent (Kamal et
al., 2000).
alcoholic liver disease is another kind of CLDs.it represents the oldest form of liver
injury known to mankind. Evidence suggests that fermented beverages existed at least
as early as the Neolithic period (cir. 10,000 BC) (OShea et al., 2010).Alcoholic liver
disease (ALD) is a major cause of chronic liver disease worldwide and in Western
countries,it presents as a broad spectrum of disorders, ranging from simple fatty liver
to more severe forms of liver injury, including alcoholic hepatitis (AH), fibrosis,
cirrhosis, and superimposed hepatocellular carcinoma (HCC) (Bin and Bataller
2011).
Non-alcoholic fatty liver disease (NAFLD) is the cause of fatty liver without
chronic excessive drinking of alcohol. The insulin resistance and/or the metabolic
syndrome are believed the principal reason of NAFLD. Obesity, diabetes type II,
hyperlipidemia and high blood pressure all contribute to NAFLD. In modern society,
over-nutrient and metabolic syndrome has been a big problem, and the NAFLD is
more and more popular in western countries (Unger and Scherer 2010).
The NAFLD may develop to non-alcoholic steatohepatitis (NASH), which
means the inflammation in liver, Similar as the CLDs mentioned above, the NASH
may progress to fibrosis and cirrhosis (Clark and Diehl 2003). The prevalence of
NAFLD is estimated around 30%. The modern gastronomic revolution encourages
people consuming excess calories by intake high sucrose soft drinks and carbohydrate
enriched fatty foods, which lead to over-nutrient and increase the risk of metabolic
syndrome. As a result, the incidence of NAFLD remarkably elevates in the world,
especially in developed countries (Milic and Stimac 2012). However, the NAFLD
morbidity in developing countries is also rises quickly in the latest decades.
] 7[
The intake of some toxic drugs will cause liver injury and CLDs. Drug induced
liver injury is the common cause of hepatitis in old patients. The antimicrobials,
central nervous system agents, immunomodulating agents , analgesics, and lipidlowering agents are the major hepatotoxic prescription medications, (Bell and
Chalasani 2009).For example, chronic hepatic inflammation and fibrogenesis have
been identified in patients who accepted long-term, low-dosage paracetamol
administration (Bonkowsky et al., 1978).
The drugs are always recognized as xenobiotics in body and metabolized in liver.
The cytochrome P-450 enzyme system in the smooth endoplasmic reticulum of
hepatocytes is responsible for the elimination of drugs. The metabolic intermediate
products may induce oxidative stress and hepatotoxicity. Drug induced liver injury
will induce chronic active hepatitis, fibrosis, primary biliary cirrhosis, steatohepatitis
and other CLDs. (Bell and Chalasani 2009).
Autoimmune liver diseases include autoimmune chronic hepatitis, primary
biliary cirrhosis and primary sclerosing cholangitis. The hepatocyte apoptosis induces
autoimmunity in liver is associated with the autoimmune diseases (Mackay 2012).
The usual hereditary hepatic diseases contain hemochromatosis and Wilson
disease, and many other CLDs are associated with the genetic factors (Thompson et
al., 2012).
These different etiologic forms of chronic liver disease (CLD) have a common
histopathological pathway that is the formation and accumulation of fibrosis leading
to the development of progressive distortion of the hepatic architecture that is the
hallmark of evolution to cirrhosis. Natural history studies indicate that advanced
fibrosis and cirrhosis develop in about 20%-40% of patients with chronic hepatitis B
or C and in a similar proportion of those with alcoholic or non alcoholic steato
hepatitis (Sebastiani and Alberti 2006).
] 8[
Biological Principals of Hepatic Fibrosis:

Several discoveries have played a critical role in understanding the pathophysiologic
basis of hepatic fibrogenesis and cirrhosis. These are discussed below (Friedman
2003 ;Rockey 2006):
1-Hepatic fibrosis initially is a wound healing response :
in which damaged regionsare encapsulated by extracellular matrix, (ECM) or scar.
In all circumstances, the characterization of the fibrotic scar is similar; specifically
the extracellular matrix proteins that make it up. The cells and soluble factors
participating in this response in liver are similar to those seen in parenchymal
injury to the kidney ( Johnson et al., 1991)., lung, and skin. This understanding
has helped to identify underlying mechanisms and may likely lead to new
therapies for fibrotic diseases of many organs, including liver.
2-The role of cellular elements in extracellular matrix production:
The discoverieshave led to the identification of cellular elements that are
responsible for extracellular matrix production and in defining how they respond
to injury. One of the most important cellular elements has been the hepatic stellate
cells.
3-The same cell type produces hepatic fibrosis regardless of the underlying
cause:
The activated hepatic stellate cell has emerged as the key cellular source of
hepatic scar. How stellate cells become activated in response to a large variety of
hepatic insults from inborn metabolic defects to chronic viral hepatitis remains
largely unknown.
] 9[
4-Soluble factors (cytokines / peptides) and cell-matrix interactions are

important mediators in wound response;
Certain soluble factors such as cytokines and peptides, aswell as extracellular
matrix (including interactions with integrins as important receptors mediating cellmatrix interactions) play a critical role in wound response.
5-The fibrotic scar, and ECM are biologically dynamic milieu, not inert
ground substances:
It has been recognized that the fibrogenic process, ECM and the fibrotic scarcan
be dynamically modulated (i.e., reversible) by way of the effect of matrix
degrading proteases and apoptosis of the effector cells.
6-Hepatic fibrosis follows chronic but not self-limited injury:
Scarring does notdevelop in patients who survive fulminant hepatitis, despite an
abundance of fibrogenic stimuli, unless chronic injury follows. The reason for this
observation is not understood, but identifying the factors that make fibrosis
reversible may provide important clues to the pathogenesis of fibrosis and
cirrhosis.
7- Fibrosis occurs earliest in regions where injury is most severe
especially inchronic inflammatory liver disease resulting from alcohol abuse or
viral infection.
8- Oxidative stress:
in chronic liver diseases (CLD) results from an increased generation of ROS (and
other reactive intermediates) and from a decreased efficiency of antioxidant
defenses. This stress contributes to excessive tissue remodeling and fibrogenesis
]10[
by stimulating the production of profibrogenic mediators by Kupffer cells and
other resident and circulating inflammatory cells. (Urtasun et al ., 2008 ).
9-Hypoxia is a critical, early fibrogenic stimulus, caused by capillarisation of
sinusoids, intrahepatic shunting, vasoconstriction, compression and thrombosis
(Rosmorduc and Housset 2010). It impairs mitochondrial function, produces
oxidative stress, induces vascular endothelial cell growth factor (VEGF) and its
receptors, and stimulates synthesis of collagen 1 in HSCs (Corpechot et al., 2002).
Hypoxia also potentiates expression of transforming growth factor, beta 1
(TGF1), (Jeong et al., 2004). contributing to both autocrine and paracrine loops
that drive angiogenesis, chemotaxis and fibrogenesis.
10- Inflammation initiates and regulates fibrosis by the release of soluble
mediators and the secretion of products that remodel/degrade the normal
basement membrane matrix within the liver (Mehal and Friedman 2007).
11- Apoptosis, a common feature of chronic liver disease, results in the
generation of apoptotic bodies, which phagocytotic clearance creates proinflammatory and fibrogenic stimuli. After engulfing the apoptotic bodies,
Kupffer cells secrete death ligands and tumor necrosis factor alpha (TNF).
Similarly, the engulfment of apoptotic bodies by HSCs triggers a profibrogenic
response with production of oxidative radicals, and upregulation of expression of
TGF1 and collagen 1 (Canbay et al., 2004 ; Zhan et al., 2006 ;Canbay et al.,
2003 ;Chakraborty et al., 2012).
HEPATIC FIBROGENESIS:
Liver fibrosis results from perpetuation of the normal wound healing response.
Repeated cycles of hepatocyte injury and repair, accompanied with inflammation and
excessive deposition of ECM proteins, result in an abnormal continuation of
]11[
fibrogenesis. Its progression depends on the etiology of liver disease and is influenced
by environmental and genetic factors (Bataller et al.,2003 ;Friedman 2003;Lee and
Friedman 2011).During hepatic fibrogenesis, significant changes in quality, quantity
and distribution of ECM components occur in the periportal and perisinusoidal space,
increasing the content of collagens and non-collagenous components (Schuppan
1990).In normal liver, the subendothelial space of Disse that separates the epithelium
(hepatocytes) from the sinusoidal endothelium, contains both an interstitial (highdensity) and a basement membranelike, perisinusoidal (low density) ECM. During
fibrosis, perisinusoidal ECM is replaced by an interstitial (scar-type) matrix with
bundles of collagen fibrils and an electron-dense basement membrane (Schuppan et
al.,2001). This is tied in with a loss of hepatocyte microvilli and a disappearance of
endothelial fenestrations. The dominant event in fibrogenic cascade is activation of
hepatic stellate cells (HSCs) (Friedman 2000), remarkably adaptable mesenchymal
cells that are vital to hepatocellular function and the livers response to injury. These
cells resident in the space of Disse are the major storage of vitamin A in the body.
They represent one-third of the nonparenchymal cells, and 15% of the total number
of resident cells in normal liver. HSCs are primary effector cells that play a pivotal
role in activating the immune response through secretion of cytokines and
chemokines and interacting with immune cells, contributing thereby to angiogenesis
and the regulation of oxidant stress, and orchestrating the deposition of ECM in
normal and fibroticdeposition of scar-tissue. These cells are generated locally, by
transdifferentiation of resident perisinusoidal HSCs and periportal fibroblasts,
(Iwaisako Ket al., 2012; Parola et al.,2008). or via the recruitment and
differentiation of bone-marrow-derived mesenchymal stem cells. (Forbes et al., 2004
; Russo et al.,2006).Activated HSCs secrete large amount of interstitial collagen,
accompanied by matrix metalloproteinases (MMPs), which induce three-dimensional
changes in the ECM. They degrade the normal basement membrane matrix, whilst
having a mild effect on the neo-matrix rich in fibrillar collagen. On the other hand,
the production of tissue inhibitor of metalloproteinase (TIMPs) by HSCs also
]12[
increases, promoting ECM accumulation by inhibiting matrix degradation, improving
survival of activated HSCs and preventing their clearance, all delaying the regression
of liver fibrosis (Han 2006;Mormone et al.,2011;Ramachandran and Iredale
2012).The changes in ECM impair the flow of metabolites between the hepatocytes
and the perfusing plasma, exposing hepatocytes to hypoxia (particularly in pericentral
zones). In unopposed fibrosis this causes distortion of hepatic architecture, formation
of septae (broad bands that divide the parenchyma into regenerative nodules),
increase in intrahepatic vascular resistance, angiogenesis and sinusoidal remodeling,
inducing portal hypertension(Thabut and Shah 2010). This ultimately leads to
pathophysiological damage and cirrhosis as the final stage (Schuppan and Afdhal
2008).Advanced fibrotic changes are strongly associated with hepatocellular
carcinoma, which further reduces chances of survival (Zhang and
Friedman
2012).Potential reversibility of hepatic fibrosis varies depending on the duration of

injury, the degree of angiogenesis, and the composition, spatial distribution, and
cellularity of scar. Cirrhotic areas resistant to degradation are fairly acellular and rich
in elastin and highly cross-linked collagen, suggesting that ECM cross-linking might
represent a point of no return in fibrosis (Ramachandran and Iredale
2012).Fibrotic patients have a good short-term prognosis, as it usually requires
several months to years of ongoing insult to reach chirrosis.(Fattovich et al., 1997).
Cirrhosis, however, as present evidence indicates, is not completely reversible.
HEPATIC STELLATE CELLS AND FIBROGENESIS:
The clarification of stellate cell responses in hepatic injury and repair has been a
significant turning point in understanding the basis of hepatic fibrosis. In particular,
the identification of stellate cell activation as a key event in fibrogenesis has provided
an important framework for conceptualizing the livers response to injury (Bataller
and Brenner 2005;Friedman et al., 2000;Friedmanet al., 2006;Gressneret
al.,2002;Kisseleva and Brenner 2006;Lotersztajn et al., 2005)
]13[
In response to liver injury, HSC undergo rapid activation, functional and

morphological changes. Activation, which occurs in two phases, initiation and
perpetuation, is, if liver injury has subsided, followed by resolution (Friedman
2004 ).
Stellate cell activation refers to the conversion of a resting vitamin A-rich cell to one
that is proliferating, fibrogenic, and contractile. While it is increasingly clear that
other mesenchymal cell populations also contribute to extracellular matrix
accumulation, stellate cell activation remains the most dominant pathway leading to
hepatic fibrosis. Moreover, stellate cell activation represents a continuum, such that
early changes in cellular phenotype may be distinct from those occurring with
progressive injury and activation in terms of growth characteristics, response to
soluble mediators, inflammatory signaling, and apoptotic potential (Dooleyet
al.,2000;Gong et al., 1998;Gressner 1996; Purps et al., 2007;Roth-Eichhorn et
al., 1999;Schnabl et al.,2003).
Activation consists of two major phases: initiation and perpetuation, followed by

resolution of fibrosis if injury subsides :
1. Initiation of HSCs activation comprises early changes in gene expression and
phenotype, which make them responsive to cytokines and other stimuli released
from affected cells, like damaged hepatocytes, bile duct cells, Kupffer cells and
inflammatory cells (Friedman and Bansal 2006 ).
2. Perpetuation results from the effects of these stimuli on maintaining the
activated phenotype and generating fibrosis. Perpetuation involves autocrine as
well as paracrine loops. It is comprised of several discrete responses including
proliferation, contractility, fibrogenesis, matrix degradation, retinoid loss, and
inflammatory cell infiltration.
]14[
3. Resolution of fibrosis refers to the fate of activated HSCs when the primary
insult is withdrawn or attenuated, and refers to pathways that cause apoptosis,
senescence, or quiescence of HSCs (Bataller and Brenner 2005 ;Kisseleva
and Brenner 2006 ; Krizhanovsky et al., 2008 ).
1. INITIATION:
The earliest changes observed during stellate activation result from paracrine
stimulation by all neighboring cell types, including sinusoidal endothelium,
Kupffer cells, hepatocytes, and platelets. As noted above, early injury to
endothelial cells stimulates production of cellular fibronectin, which has an
activating effect on stellate cells (Jarnagin et al., 1994).
Endothelial cells are also likely to participate in conversion of TGF- from
the latent to active, profibrogenic form.
Platelets are another important source of paracrine stimuli, including PDGF,
TGF- ,and EGF( Bachem et al.,1989).
Kupffer cell infiltration and activation also contributeto stellate cell
activation. Kupffer cells stimulate matrix synthesis, cell proliferation, and release
of retinoids by stellate cells through the actions of cytokines (especially TGF-)
and reactive oxygen intermediates/lipid peroxides (Bilzer et al., 2006).
Hepatocytes are a potent source of fibrogenic lipid peroxides, although
effects on stellate cell collagen synthesis and proliferation may be dose dependent
(Novo et al., 2006). Hepatocyte apoptosis following injury also promotes stellate
cell initiation through a process mediated by Fas(Canbay et al., 2004;Canbay et
al., 2002). This process may involve the TNF-related apoptosis inducing ligand
(TRAIL) (Canbay et al., 2002).
Whereas hepatocyte necrosis associated with lipid peroxidation is considered a
classical inflammatory and fibrogenic stimulus, recent findings also implicate
apoptosis, or programmed cell death, in the fibrogenic response. Apoptotic
fragments released from hepatocytes are fibrogenic towards cultured stellate cells
]15[
(Canbay et al.,2003). and activate Kupffer cells (Canbay et al., 2003). Also, Fasmediated hepatocyte apoptosis is fibrogenic in vivo in experimental animals
(Canbay A et al., 2002).
2. PERPETUATION
Perpetuation of stellate cell activation involves at least seven discrete
changes
in
cell
behavior:
proliferation,
chemotaxis,fibrogenesis,
contractility,matrix degradation,retinoid loss,and WBC chemoattractant/cytokine

release. The net effect of these changes is to increase accumulation of extracellular
matrix. As an example, proliferation and chemotaxis lead to increased numbers of
collagen-producing cells. Cytokine release by stellate cells can amplify the
inflammatory and fibrogenic tissue responses, and matrix proteases may hasten the
replacement of normal matrix with one typical of the wound scar. (Vandana et
al.,2014).
PROLIFERATION:
PDGF is the most potent stellate cell mitogen identified (Pinzani 2002).
Induction of PDGF receptors early in stellate cell activation increases
responsiveness to this potent mitogen (Wong et al., 1994).
CHEMOTAXIS:
Stellate cells can migrate towards cytokine chemoattractants (Marra
2002;Pinzani and Marra 2001), explaining in part why stellate cells align within
inflammatory septae in vivo. A number of chemoattractants have been identified,
prominent among which are PDGF(Ikeda et al.,1999;Kinnman et al., 2000).
MCP-1 (Marra et al., 1999), and CXCR3(Bonacchi et al., 2001).In contrast,
adenosine (Hashmi et al., 2007). blunts chemotaxis and may immobilize cells
once they reach the site of injury.
FIBROGENESIS:
]16[
Stellate cells generate fibrosis not only by increased cell numbers, but also
by increasing matrix production per cell. The best-studied component of hepatic
scar is collagen type I, The most potent stimulus for production of collagen I and
other matrix constituents by stellate cells is TGF-,which is derived from both
paracrine and autocrinesources (Breitkopf et al., 2006;Inagaki et al., 2007).TGF also stimulates the production of other matrix components including cellular
fibronectin and proteoglycans (George et al., 2000).
CONTRACTILITY:
Contractility of stellate cells may be a major determinant of early and late
increases in portal resistance during liver fibrosis. The collagenous bands typical
of end-stage cirrhosis contain large numbers of activated stellate cells. These
impede portal blood flow by constricting individual sinusoids and by contracting
the cirrhotic liver(Vandana et al.,2014).
MATRIX DEGRADATION:
Fibrosis reflects a balance between matrix production and degradation. The
degradation of extracellular matrix is a key event in hepatic fibrosis. Early
disruption of the normal hepatic matrix by matrix-degrading proteases hastens its
replacement by scar matrix, which has deleterious effects on cell function.
Disruption of the normal liver matrix is also a requirement for tumor invasion and
desmoplasia and may be particularly relevant to pancreatic stellate cells.
Degradation in these contexts is referred to as being pathological. On the other
hand, resorption of excess matrix in patients with chronic liver disease provides
the opportunity to reverse hepatic dysfunction and portal hypertension (Vandana
et al.,2014).
RETINOID LOSS:
]17[
Activation of stellate cells is accompanied by the loss of the characteristic
perinuclear retinoid (vitamin A) droplets. Whether retinoid loss is required for
stellate cells to activate, and which retinoids might accelerate or prevent activation
are not clarified (Vandana et al.,2014).
Fibrosis Regression:
Studies on rodent models have demonstrated that, once a liver injury is eliminated,
fibrosis regression occurs. The restoration of fibrinolytic activities, such as the upregulation of MMPs and down-regulation of TIMPs, drives the reversal of fibrosis
The clearance of activated HSCs or myofibroblast through apoptosis (Iredale et al.,
1998). senescence (Krizhanovsky et al., 2008).or reversion into quiescent
phenotypes is also crucial in fibrosis resolution (Kisseleva et al.,2012;Troeger et
al.,2012).
]18[
A key study (Hammel et al., 2001), described a group of patients who underwent
surgical decompression of an obstructed biliary system. In some patients, liver
fibrosis significantly regressed after decompression, implying that fibrosis caused by
biliary obstruction is reversible in some instances. An important feature of this study
was that 19 of the 22 liver biopsies were large wedge specimens taken from the third
segment of the left hepatic lobe under direct vision at the time of surgery, thereby
negating concerns regarding sampling variation. Fibrosis regression has also been
documented in patients with hepatitis B(Dienstag et al., 2003). hepatitis C (Poynard
et al., 2002). delta hepatitis (Lau et al., 1999).and immunosuppressive therapy for
autoimmune hepatitis (Dufour et al., 1997). Chronic HCV infection is the most
extensively studied condition, and therapy (IFN-plus ribavirin) with viral clearance
results in fibrosis improvement. Importantly, nearly half of patients with cirrhosis
exhibit reversal of fibrosis to a significant degree. Therefore studies that used
pathological specimens and paired biopsies from trials of antiviral treatments in
chronic hepatitis have shown that matrix degradation occurs in advanced human
cirrhosis. Nevertheless, incontrovertible evidence for complete spontaneous reversal
of advanced cirrhosis does not exist. Moreover, experimental models in rodents
indicate that aspects of fibrosis in advanced cirrhosis are truly irreversible ,As well as
supporting the findings of fibrosis reversibility in humans, rodent models in which
spontaneous recovery from liver fibrosis and cirrhosis occurs have allowed the indepth analysis that is needed to identify the critical features of this process (Iredale
et al.,1998;Murphy et al.,2002;Issa et al.,2004). HSCs, other liver myofibroblasts
and inflammatory cells involved in the fibrotic process, including macrophages and
Kupffer cells, secrete a repertoire of matrix-degrading MMPs (Benyon and Arthur
2001).These enzymes degrade collagen and other matrix molecules, and their
presence in the fibrotic liver highlights the potential dynamic nature of scarring
within the liver. Molecular studies of the expression of mRNA for these enzymes
(including those with collagenolytic activity) have demonstrated that they are
]19[
expressed in the liver even in cirrhosis, but their activity is limited by powerful
inhibitors, TIMP-1 and TIMP-2 (Benyon et al.,1996). In recovery, expression of
TIMP-1 and TIMP-2 decreases rapidly while matrix-degrading MMPs continue to be
expressed, resulting in increased collagenase activity and consequent matrix
degradation within the liver.
Liver fibrosis scoring/staging

Evaluating Liver Fibrosis:
Schematic diagram of noninvasive methods used to assess liver fibrosis and
cirrhosis(Ahmad et al., 2011).(figure
Figure( )
Importance of liver fibrosis staging:

]20[
Measurement of fibrosis not only helps to stage the severity of disease, it allows serial
determination of disease progression. The level of fibrosis may play an important role
in clinical management and determine patients prognosis. For example, aggressive
therapy is more appropriate in HCV-infected patients with advanced fibrosis. Further,
the fibrosis progression rate is an important predictor of the time to develop cirrhosis
(Poynard et al., 2001).
Assessment of fibrosis stage and rate of fibrosis progression can be valuable for at
least three reasons:
(1)the actual stage of fibrosis will indicate the likelihood of response to a-interferon
or ainterferon/ ribavirin, as the advanced stages of fibrosis (F3 or F4) generally have a
lower response rate to antiviral therapy; (Wright 2002;Arenas and Vargas 2004).
(2) if little fibrosis progression has occurred over a long interval, then treatment with
antiviral therapy may be deemed to be less urgent and it may be safe to await more
effective and/or better-tolerated therapy;
(3) the approximate time to the development of cirrhosis can be estimated. This
would not, however, indicate if or when clinical liver failure would occur, as the
complications of liver disease may be delayed for up to a decade or more after the
establishment of cirrhosis. As genetic risk markers that predict a rapid fibrosis
progression rate are developed, this information, combined with the absolute stage of
fibrosis, may enable more accurate identification of patients at risk for disease and
thus in need of antifibrotic therapy.
Liver fibrosis evaluation methods
They can be divided into
]21[
(1) Invasive:
Liver Biopsy and Histologic Assessment of the Liver
Liver biopsy is considered the gold standard for diagnosing and assessing liver
fibrosis. The liver biopsy provides information on both the grade (degree of
inflammation that reflects ongoing liver disease injury) and the stage (amount of
currently established fibrosis).
Liver biopsy the gold standard for staging of liver fibrosis:
Liver biopsy was first described by Paul Ehrlich, 130 years ago, as a diagnostic tool
of the chronic liver disease. For the past 50 years liver biopsy has been considered to
be the gold standard for staging of liver fibrosis(Sebastiani and Alberti 2006).
It can be performed using blind percutaneous technique, computer tomography or
ultrasound-guided, transjugular, open surgical, laparoscopic and even endoscopic
approaches. Microscopic examination of liver tissue often provides the definitive
diagnosis and leads to effective management of the liver disorders. Because fibrosis is
tissue damage, biopsy is by definition the only direct tool to assesses liver fibrosis
(Braticevici et al., 2011).
In the majority of liver centers worldwide, liver biopsy is performed as a blind or
ultrasound-guided puncture, as either an out- or in-patient procedure. This procedure
allows physicians to obtain diagnostic information not only on fibrosis, but also on
many other liver injuring processes, such as inflammation, necrosis, steatosis, hepatic
deposits of iron or copper (Sebastiani and Alberti 2006).
Liver punctures are considered to be relatively safe procedures with complication
rates ranging from 0.75% up to 13.6%
(Myers et al.,2008).The most frequent
complications are minor bleeding or pain. After efficient substitution with clotting
factors, percutaneous liver biopsy is also possible in patients with inherited bleeding
disorders with no obvious increase in complication rates (DiMichele 2003, Schwarz
2008). (Schwarz et al.,2008).
]22[
Transjugular puncture of the liver via cannulation of a hepatic vein is an alternative,
which can be performed in patients with severe coagulation deficiencies. It is resource
intensive and carries a risk of intrahepatic hemorrhage or capsule perforation with
intra-abdominal bleeding. (Mammen et al., 2008 ;Kalambokis et al.,2007).
Laparoscopy and mini-laparoscopy are even more invasive procedures for obtaining
liver biopsy. A recent randomized trial showed a higher detection rate of liver
cirrhosis as compared to percutaneous biopsies with lower complication rates for
laparoscopy (Denzer et al.,2007 ; Helmreich-Becker et al.,2003).
DISADVANTAGES OF LIVER BIOPSY:
The quality and reliability of fibrosis staging via histopathological assessment of liver
biopsy specimens depends largely on the size of the specimen and the number of
portal fields. The biopsy should be 20-25 mm long and more than 11 portal tracts
should be visible (Bedossa 2003, Cholongitas 2006, Rousselet 2005). (Bedossa et
al.,2003),(Cholongitas et al.,2006), (Rousselet et al., 2005).However, in daily
practice these requirements may not be easy to achieve; and even if a large enough
biopsy is acquired, the specimen only reflects about 1/50,000 of the whole liver. Thus,
liver biopsies are particularly prone to sampling errors and may like non-invasive
markers have difficulties in discriminating between adjacent stages of fibrosis (i.e.,
F1 vs. F2 or F2 vs. F3) ,also high cost, and inaccuracy due to inter- and intraobserver variability of pathologic interpretations (Sebastiani and Alberti 2006).
In addition, there is a small but important risk of liver biopsy-associated morbidity
and mortality; pain, hypotension, intraperitoneal bleeding and injury to the biliary
system. The risk for hospitalization after liver biopsy is 1-5%, the risk for severe
complications is 0.57%, and mortality rate is 0.009-0.12%. Because of these reasons,
some patients may choose to go without liver biopsy.(Terjung et al., 2003);West and
Card 2010).
]23[
Classification of Liver Histology

The history of the fibrosis scoring systems dates back to 1981 when the histological
features of chronic hepatitis were evaluated for potential importance in determining
its prognosis by Knodell and colleagues(Knodell et al.,1981).
Several histologic scoring systems have been developed tograde (inflammation) and
stage (fibrosis) hepatic disease caused by hepatitis. Some scoring systemsare
complex, such as Knodell or Ishak (revised Knodell system), and because of their
complexity are primarily only used in large cohort clinical trials. For general clinical
purposes, most use the less complicated scoring systems that have only three to four
categories, such as Batts and Ludwig, METAVIR, and International Association for
Study of the Liver (IASL).The Ishak score, or revised Knodell system , considers
grading and staging as two separate items; liver fibrosis is classified as: 0 = absent, 12 = mild, 3-4 = moderate and 5-6 = severe/cirrhosis. The grading system has been
subsequently modified by other pathologists (Baranova et al.,2011)
The Metavir classification system includes two separate scores, one for
necroinflammatory grade (A) and another for the fibrosis stage (F). The grade is
usually scored from 0-4, with A0 being no activity and A3 to A4 considered severe
activity. The stage scored from F0 to F4 .F0 is the absence of fibrosis, F1 is portal
fibrosis without septa (MILD FIBROSIS), F2 is portal fibrosis with rare
septa(MODERATE FIBROSIS), F3 is numerous septa without cirrhosis(BRIDGING
FIBROSIS), and F4 is cirrhosis .the METAVIR score is considered best in HCV
fibrosis(Bedossa and Poynard 1996).
Non-invasive:
]24[
Liver histology, by helping visualise the fibrosis, has been considered the gold
standard for assessment and measurement of progression of fibrosis. However, liver
biopsy is an invasive procedure, unsuitable for tight monitoring. In addition,
fragmented or small-sized specimens may cause the underestimation of fibrosis.the
disadvantages of this method have motivated researchers and clinicians to look into
more non-invasive strategies. These strategies are based either on single serum
markers, compositional scores derived from combinations of different markers, or
modifications of imaging techniques (Coco B et al., 2007).
Serum markers:
Serological markers refer to the measurement of one or more molecules within blood
or serum correlating to hepatic fibrosis (Friedman 2000;Friedman 2003;Kelleher
and Afdhal 2005). Among serum markers, there are direct serum markers,
reflecting either the deposition or the removal of extra-cellular matrix in the liver,
such as glycoproteins, the collagens family and collagenases and their inhibitors for
which hyaluronate has been the most studied. There are indirect serum markers of
fibrosis or simple routine blood tests such as prothrombin index, platelet count, and
AST/ALT ratio. Apart from hyaluronate, direct and indirect markers, when used
individually, are useful for the diagnosis or the exclusion of cirrhosis but have limited
accuracy for the diagnosis of significant fibrosis. Their levels vary by changes in their
clearance, metabolism, and excretion,and their significant contribution from nonhepatic sources, such as, bones, joints, lungs, kidneys and skin(Idobe et al.,1998;Saif
et al.,2005). More sophisticated indices combining panels of indirect and direct
markers, for which the pionniers have been the Fibrotest (Biopredictive, Paris,
France) and the AST to Platelet Ratio Index (APRI), have been proposed. Five indices
are protected by patents and are currently commercially available: the Fibrotest in
Europe (Biopredictive, Paris, France) or Fibrosure in the USA (LabCorp,
Burlington, NC, USA), the Fibrometers (BioLiveScale, Angers, France), the
FibroSpect II (Promotheus Laboratory Inc. San Diego, Ca, USA), the ELF
]25[
(Enhanced Liver Fibrosis Test, iQur Ltd, Southampton, UK) and the Hepascore
(PathWest, University of Western Australia, Australia). To date, FibroTest and APRI
have been the most extensively studied (mainly in hepatitis C) (Castera 2010).
Direct serum markers:
Direct markers which can reflect extracellular matrix deposition (e.g; Procollagen I
peptide, Procollagen III peptide, Type I collagen, Type IV collagen, Laminin and
Hyaluronic acid) or degradation, (e.g.; MMP-2, TIMP-1, -2) and some cytokine (e.g.;
TGF-beta, TGF-alpha and PDGF) have been used widely to assess liver fibrosis with
good reproducibility (Mohamed 2015).
Transforming growth factor-1 (TGF- 1):
It is a cytokine involved in tissue growth, differentiation, ECM components
production and the immune response. TGF-1 is commonly accepted as a central
component of fibrogenic response to wounding and is up-regulated in a variety of
different diseases. A correlation between TGF-1 levels and the rate of fibrosis
progression is widely accepted(Baranova et al., 2011).
Connective tissue growth factor (CTGF):
It is synthesized in response to profibrogenic factor TGF- by both activated HSC
and hepatocytes. However, serum CTGF levels decrease in the end-stage cirrhosis
(Gressner and Gressner 2008).
Extracellular matrix components:
Procollagen peptides:
Procollagen type I carboxy-terminal peptide (PICP):
levels are normal in patients with mild chronic hepatitis C (CHC) and elevated in
50% of patients with moderately advanced or advanced chronic hepatitis C, including
patients with liver cirrhosis of this etiology (Jarcuska et al.,2010).
]26[
Procollagen type III amino-terminal peptide (PIIINP):

is a product of cleavage of procollagen and has been proposed as a serum marker of
hepatic fibrosis more than two decades ago (Rohde et al., 1979).
Its relative
concentration in the basement membrane is higher in hepatic fibrogenesis (Veidal et

al.,2010). In several studies in patients with chronic HCV infection, this biomarker
showed
only
moderate
diagnostic
accuracy(Stauber
and
Lackner
2007).Unfortunately, PIIINP is not specific for the fibrosis of the liver as it is also
elevated in acromegaly, lung fibrosis, chronic pancreatitis, and rheumatologic disease.
(Fontana and Lok 2002).
Hyaluronic acid (HA):
HA is a polysaccharide present in ECM and elevated in serum in patients with hepatic
fibrosis. Commercial test kits are available from Corgenix (Westminster, Colorado).
The diagnostic accuracy of HA was found to be superior to that of PIIINP (Guechot
et al.,1996).
Matrix metalloproteinases and their inhibitors:

Matrix metalloproteinases (MMPs):
Excess ECMP are degraded by matrix metalloproteinases which are in turn inhibited
by tissue inhibitors of metalloproteinases (TIMPs). The three most commonly studied
MMPs are MMP-2, MMP-3, and MMP-9. MMP-2 is secreted by activated HSCs
(Takahara et al., 1997).
Tissue inhibitors of metalloproteinases:
TIMP-1 controls activity of most MMPs, whereas TIMP-2 specifically inhibits MMP2. TIMPs-dependent inhibition of ECMP degradation may promote liver fibrosis;
elevation of TIMPs levels has been observed in chronic liver disease. For example,
]27[
CHC causes the elevation of both TIMP-1 and TIMP-2 in corollary with fibrosis
progression (Walsh et al.,1999).
Paraoxonase 1 (PON-1):
It is an enzyme that hydrolyzes lipid peroxides, has antioxidant properties and
influences hepatic cell apoptosis. Measurement of serum PON-1 activity has been
proposed as a potential test for the evaluation of liver function, however, its clinical
acceptance is limited due to instability and toxicity of its substrate, paraoxon (Camps
et al.,2009).
Laminin:
It is a major non-collagenous glycoprotein synthesized by the HSC and deposited in
the basement membrane of the liver. During fibrosis, laminin accumulates around the
vessels, in the perisinusoidal spaces and near the portal tract (Kropf et al.,1988)
Combined direct markers:

FibroSpect :
FibroSpect assay is a combination of threeparameters: HA, TIMP-1 and alpha-2macroglobulin and can differentiate between no/mild and moderate/severe fibrosis.
(Patel et al., 2004 ; Zaman et al.,2007).
The ELF test:
combines three direct serum markers; hyaluronic acid (HA), procollagen III amino
terminal peptide (PIIINP) and tissue inhibitor of metalloproteinase 1 (TIMP-1) using
an algorithm developed by the European Liver Fibrosis Group based on an
international multi-centre cohort study in 1,021 patients1.
(Rosenberg et al., 2004).
]28[
Indirect serum fibrosis markers:
Initial screening with simple laboratory tests, such as platelet count, prothrombin
time, albumin, total bilirubin, alpha-2-macroglobulin, and haptoglobin and serum
aminotransferase level are commonly performed in clinical practice in an attempt to
estimate fibrosis and identify overt cirrhosis and are easily available. although cannot
reflect changes in extracellular matrix activity; they can reflect changes in liver
function .
Serum ALT, AST and AFP levels:

Serum ALT released from liver tissue into the circulation in proportion to the degree
of hepatocellular damage due to viral infections and toxic substances (Daxboeck et
al.,2005).
ALT is thought as one of the more sensitive marker of liver injury and disease
progression(Sherman 1991;Dufour et al.,2000;Akkaya et al.,2007).However, ALT
enzymatic activity may not always reflect the degree of hepatic damage as about 26%
patients have persistently normal ALT levels but have a histological score greater than
A1F1(Kim et al.,2009). Serum AST levels are most important predictor of
histological activity than ALT (Shiffman et al.,2006 ;Okuda et al.,2002; Zechini et
al.,2004).
Serum AFP is alpha-1-globulin secreted by fetal hepatocytes and fetal gastrointestinal
tract. Elevated serum AFP levels are associated with acute and chronic HCV, toxic
liver injury concentrations and correlate with tumor size and decrease or normalize
after tumor removal. Elevated AFP levels are observed in cirrhotic patients (Cedrone
et al., 2000 ;Chu et al.,2001;Chen et al.,2007;Tamura et al.,2009).
]29[
Prothrombin time (PT):
PT reflects the synthesis capacity of the liver and essential mechanism of blood
coagulation. Its clinical referencerange is usually around 12-15 seconds. Prolonged
PT is associated with esophageal varices and is one of the earliest indicators of liver
cirrhosis (Croquet et al., 2002; Pilette et al.,1999;Craxi et al.,2000).
Combined indirect markers:

AST/ALT ratio (AAR):
Sheth et al. reported an AST/ALT ratio 1 having 100% PPV for the presence of
cirrhosis in chronic HCV patients(Sheth et al.,1998). Reedy et al. observed that AAR
failed to predict cirrhosis accurately in HCV patients (Reedy and 1998), while
Giannini et al. reported high diagnostic accuracy of the AAR for prediction of
cirrhosis in HCV infected patients (Giannini et al., 2003).However, many authors
could not able to find high accuracy of this marker (Afdhal and Nunes 2004
;Lackner et al.,2005 ;Fujii et al., 2009).
Age-AST model:
It is calculated as: Age-AST model = Age (year)/AST (IU/L)(Liu et al., 2012).
Platelet count:
Hepatic fibrosis may lead to thrombocytopenia as a consequence of impaired
synthesis of thrombopoietin and/or sequestering ofplatelets in an enlarged spleen.
Surprisingly, few data exist on the diagnostic value of platelet count per se although
the platelet count has been included in several composite fibrosis scores. Ono et al.
(1999) reported the use of platelet count could discriminate F4 from F1-F3 in 75%80% of patients with CHC,(Ono et al.,1999).In Lackner et al. (2005) study, a platelet
count of < 150 109/L had a positive predictive value (PPV) > 90% for significant
]30[
fibrosis, whereas at a cut-off of 150 109/L it had a negative predictive value
(NPV) > 90% for cirrhosis.(Lackner et al.,2005).
Age- platelet index (API):
It is calculated as API = Age (year)/PLT ( 109/L)(Poynard and Bedossa 1997).
Cirrhosis discriminant score (CDS):
Platelet count has been also combined with AAR and prothrombin time in the CDS
but the diagnostic accuracy of these composite scores was not superior to platelet
count per se (Bonacini et al., 1997;Lackner et al.,2005).
Globulin/platelet (GP) model:
It is calculated as: GP model = GLOB (g/mL) 100/PLT ( 109/L). Using this model
chronic HBV-infected patients with minimal fibrosis and cirrhosis can be diagnosed
accurately, and the clinical application of this model may reduce the need for liver
biopsy in HBVinfected patients (Liu et al.,2012).
AST to platelet ratio Index (APRI):
APRI was the simplest and accurate test for significant liver fibrosis and cirrhosis
(Wai et al.,2003). Several authors verified this marker for fibrosis and cirrhosis and
found it better than AAR. However, APRI was unable to identify individual stages of
fibrosis (Fujii et al.,2009 ;Poynard et al.,2007;Cales et al.,2005;Bourliere et
al.,2006;Parise et al.,2006; de Ledinghen et al.,2006;Halfon et al.,2007;Leroy et
al.,2008;Cales et al.,2008;Kamphues et al., 2010).
AST to platelet ratio index (APRI):
It is calculated as: APRI = [(AST/Upper limit of normal)/platelet count (109/L)]
100. Its diagnostic accuracy for both significant fibrosis and cirrhosis has been
confirmed. Using the cut-offs proposed by Wai et al approximately 50% of the
patients can be correctly classified without a liver biopsy (Wai et al.,2003).
]31[
King's score:
It is calculated as: King's score = age (years)xAST (IU/L)xINR/platelet count (109/L)
. It is a simple and accurate index for predicting cirrhosis in chronic hepatitis C. The
AUROC for predicting cirrhosis and significant fibrosis (F3-6) were 0.91 and 0.79,
respectivelyCrosset al.,2000).
PGA index:
It combines the measurement of the prothrombin index, - glutamyl transferase levels
and apolipoprotein A1. It was subsequently modified to the PGAA index by the
addition of 2-macroglobulin, which resulted in marginal improvement in its
performance. However, overall accuracy of this index is relatively low.
(Lu et al.,2003;Nguyen-Khac et al.,2008).
FibroTest/FibroSure:
FibroTest is the combination of five markers: alpha-2- macroglobulin, haptoglobin,
apolipoprotein A1, GGT and total bilirubin (Imbert-Bismut et al.,2001;Bourliere et
al., 2006). The serum levels of haptoglobin and apolipoprotein A can be affected by
conditions unrelated to CLD(Corona et al., 2010).
ActiTest:
ActiTest reflects both necroinflamatory activity and liver cirrhosis. It is modified
form of Fibrotest with addition of ALT level (Imbert-Bismut et al.,2001).
Fibrotest (Fibrosure):
French investigators analyzed an extensive array of biochemical tests in 339 patients
with CHC and identified a panel of 5 markers which could best predict the stage of
fibrosis: 2-macroglobulin, haptoglobin, apolipoprotein A1, GGT, and total bilirubin
(Imbert-Bismut et al.,2001).
]32[
FibroSure, FibroTest-ActiTest:
The HCV-FibroSure and the FibroTest-ActiTest are two identical tests marketed as
FibrotestTM in Europe and as FibrosureTM in the United States (Stauber and
Lackner 2007).
Fibro Index:
Proposed by Japanese investigators It combines three markers; AST, platelet count
and gamma globulin. not an adequate tool to be used alone but may serve as an
adjunct along with other fibrosis markers ( Koda et al.,2007).
Fibro-quotient (FibroQ) index:

It includes prothrombin time international normalized ratio (INR), platelet count,
AST, ALT, and age, and it is calculated as FibroQ = 10 (age AST INR)/(ALT
platelet count) to predict significant fibrosis (Hsieh et al.,2009).
Forns Index:
The Forns Index uses simply obtained parametersage, gamma-glutamyltransferase
(GGT), cholesterol, and platelet countbut it requires a relatively complicated
calculation (Forns et al.,2002).
SteatoTest:
It incorporates the FibroTest, ALT, body mass index, serum cholesterol, triglycerides
and glucose adjusted for age and gender. It has 63% PPV for steatosis prevalence with
93% NPV(Poynard et al.,2005).
Model 3:
]33[
This model is based on AST, platelet count and prothrombin time expressed as
international normalized ration (INR). Patients with liver cirrhosis can be excluded at
cutoff value of < 0.20 with 99% NPV(Lok et al., 2005 ;Cheong et al.,2010).
Fibrosis Index:
This index comprises of platelet count and albumin contents. It can differentiate
significant fibrosis and cirrhosis from mild fibrosis (Ohta et al., 2006).
Phol Score:
This index comprises of AST, ALT and platelet count. It showed great accuracy for
discriminating significant fibrosis and cirrhosis with high PPV and NPV. However, it
showed limited ability to predict fibrosis in later study(Pohl et al., 2001 ;Cheung et
al., 2008).
Bonacini Index:
This index incorporates ALT/AST ratio, INR and platelet count. It showed 94%
specificity for predicting significant fibrosis in initial cohort (Bonacini et al.,1997).
Indirect serum markers are easily available and routinely used. These markers have
the ability to differentiate fibrosis and cirrhosis but lesser extent to direct serum
markers. APRI and FibroTest are most validated serum markers (Ahmad et al.,2011).
Composite fibrosis markers:

Direct and indirect markers may be used alone or in combination to produce
composite scores which can be relatively simple to calculate or based on complex
formulas (Soresi 2014).
FibroMeter:
]34[
mcan differentiate fibrosis progression in viral disease consist of combination of
HA, AST, platelet count, prothrombin index, alpha-2-macroglobulin, urea and age of
the patients. Cales et al. (2005) reported superior diagnostic accuracy of Fibrometer to
Forns index, Fibrotest, and APRI (Cales et al.,2005) However, this finding was not
confirmed in an external validation study (Halfon et al., 2007 ;Cales et al.,2010).
Liver Score:
This index was formulated using six variables including age, ALT, GGT,
apolipoprotein, A2M and HA. The index was considered to be able to discriminate
various fibrosis stages, with negative predictive value (NPV) of 83% and PPV of
89%. The sample size, however, was small and the study mentioned that results
needed to be validated(Arain et al.,2011).
Hepascore also known as a FibroScore:
was developed by Australian investigators and consists of bilirubin, GGT, HA,alpha2-macroglobulin, gender and age. AUROC of this test is 0.85, 0.96 and 0.94 for
significant fibrosis, advanced fibrosis and cirrhosis, respectively (Adams et
al.,2005;Leroy etal., 2007;Becker et al.,2009;Guechot et al., 2010).
Forns Score:
This score is based on assay of age, GGT, platelets and cholesterol (Forns et
al.,2012).
Fibrospect II (FSII):
This is an assay of three serum markers HA, TIMP-1, A2M which are extracellular
matrix markers (Guajardo-Salinas and Hilmy 2010).
Fibrometer:
]35[
This is a non-invasive test which links platelets, prothrombin index, aspartate
aminotransferase, A2M, hyaluronate, urea, and age. It indicates the amount of fibrosis
as percentage of total liver mass (Leroy et al.,2014).
Shasta Index:
It combines HA, AST and albumin. Optimal results of this assay are observed in
extreme conditions. This assay showed similar accuracy with FibroTest (Kelleher et
al.,2005).
Apricot (FIB-4):
This assay combines four markers: AST, ALT, platelet count and age. This index can
predict significant fibrosis in patients infected with HIV/HCV. Later studies validated
this index not only in co-infected patients but also in HCV infected patients (Sterling
et al., 2006 ;Cales et al.,2008 ;Vallet-Pichard et al.,2007 ;Mallet et al.,2009).
Fibrosis-cirrhosis index (FCI):
FCI = (ALPxbilirubin)/(albuminxplatelet count). The FCI accurately predicted
fibrosis stages in HCV infected patients and seems more efficient than AAR, APRI,
FI and FIB-4 used serum indexes (Ahmad et al.,2011).
Sud Index:
This assay is also known as FPI comprises of age, AST, cholesterol, insulin resistance
and alcohol intake. This index showed high specificity and PPV for detecting advance
fibrosis(Et al.,2004).
Testa Index:
This index relate platelet count and spleen diameter. This ratio showed 78%
concordance with the histological score (Testa et al.,2006).
]36[
FIB-4, Fibrometer and Hepa- score are most precise and validated serum markers.
Combined serum markers are easily available and most preferable non invasive serum
markers now a day (Ahmad et al.,2011).
Others markers for liver fibrosis evaluation:
C-Caffeine Breath Test (CBT):
Caffeine has high oral bioavailability and undergoes hepatic metabolism and can be
use as quantitative test for liver function (Desmond et al.,1980).
Park et al. performed caffeine breath test (CBT) and observed the correlation of
orally administrated caffeine with plasma caffeine clearance and degree of liver
dysfunction. Chronic patients showed significantly reduced CBT values when
compared with controls (Park et al.,2003).
Differentially expressed proteins:
Differentially expressed proteins were identified by mass spectroscopy among
different degrees of fibrosis (F0-F4) and between early (F0-F1) and late (F2-F4)
fibrosis. Mac-2-binding protein, alpha-2-macroglobulin and hemopexin levels were
found increased while A-1-antitrypsin, leucine-rich alpha-2-glycoprotein and fetuin-A
were decreased in advanced fibrosis F4 as compared to early fibrosis F0/F1 (Cheung
et al.,2008).
Predicted liver fibrosis score (PLF score):
It is derived from Fibroscan and multiple serological tests. It is calculated as: PLF
score = 0.956 + 0.084 TE - 0.004 King score + 0.124 Forns score + 0.202
APRI score (Bota et al.,2011).
Recent serum fibrosis markers
Serum bile acid (SBA) model:
]37[
Halfon et al. (2013) analyzed SBA levels of 135 patients with CHC infection and
correlated these levels with the degree of liver fibrosis determined by liver biopsy.
They assessed the accuracy of SBA levels as predictor of liver fibrosis by comparison
to patients Fibrotest scores. The SBA levels were significantly higher in patients with
severe Fibrosis (Metavir F3-F4) compared to non-severe fibrosis (Metavir F0-F2).
Furthermore, the ROC curve based on a new model that included serum bile acids,
age, BMI, serum AST, glucose and cholesterol levels suggested that this combination
reliably predicts the degree of liver fibrosis with high degree of accuracy, and is not
inferior to the Fibrotest score(Halfon et al.,2013).
Carbohydrate 19.9 antigen (CA19.9):
CA19.9 is a glycoprotein expressed by several epithelial cancers, as well as in normal
pancreatic and biliary duct epithelia, and it is used currently in the diagnosis and
follow-up of gastrointestinal tumors. In a study of Bertino et al. (2013) on 180
patients, 116 with HCV-related chronic liver disease and 64 with HBV-related chronic
liver disease, they suggested that elevation of CA 19.9 is related to the severity of
fibrosis and to the viral etiology of hepatitis. They proposed that CA19.9 could be
used in the combinations with the other markers already in use, in order to increase
the diagnostic accuracy of the available tests, rising both PPV and NPV predictive
values (Bertino et al.,2013).
Limitations of fibrosis serum biomarkers
there are many limitations for serum biomarkers. They are not liver-specific and have
a tendency to be more elevated in the presence of inflammation. In addition, serum
marker readings may be falsely high due to their low clearance rates, which are
influenced by dysfunction of endothelial cells, impaired biliary excretion or renal
function. Until now, most serum biomarkers have only been used as investigative,
rather than diagnostic, parameters in the clinic (Grigorescu 2006).
]38[
From the whole range of surrogate markers only a few are in clinical use. The simple
APRI score has been widely studied in HCV and HBV as well as in coinfected
patients. using this marker alone, only about one third of all biopsies can be avoided.
Fibrotest has also achieved some clinical significance. However, this test may not be
available for all patients (Cacoub 2008; Lebensztejn 2005; Vallet-Pichard 2008;
Wai2006).
Imaging/scanning techniques
Imaging techniques are rational noninvasive approach to assess liver fibrosis.
Imaging techniques are not only capable to detect changes in the hepatic parenchyma,
these can differentiate between moderate and severe fibrosis. However, high cost and
lack of validation of concerning studies remains controversial(Ahmad et al.,2011).
Ultrasonography (US):
Ultrasound can potentially identify various factors that are useful in evaluating
chronic liver disease: nodularity of the liver surface (which reflects the presence of
regenerative nodules and fibrous septa often seen in cirrhosis), coarseness of the
parenchyma, size of lymph nodes around the hepatic artery, patency and flow of veins
and arteries, spleen size (which if enlarged can suggest portal hypertension),
hepatocellular carcinoma, and small volume ascites.. A number of studies proposed
the role of ultrasonography as a non-invasive diagnostic marker of liver fibrosis and
revealed a great sensitivity of ultrasonography to detect late stages of progressive
hepatic fibrosis, but a limited capability to measure mild or moderate fibrosis(Aube
et al.,1999 ;Mathiesen et al.,2002 ;Colli et al., 2003 ;Zheng et al., 2003 ;Colli et
al., 2005 ;Shen et al.,2006).
]39[
Transient Elastography (FibroScan):

an applicable alternative to liver biopsy Transient elastography measures tissue
stiffness. It can measure liver sample size 100 times greater than a standard biopsy
sample size, as liver biopsy size strongly effects the grading of chronic viral hepatitis.
(Sandrin et al.,2002 ;Sandrin et al.,2003 ;Colloredo et al., 2003 ;Cobbold et al.,
2007).
FibroScan results reported 100% sensitivity and specificity for PPV & NPV
respectively.In a study of 935 patients Fibroscan was found to be 97% successful in
grading chronic HCV infection (Colletta et al.,2005).In another study on 711
patients, liver stiffness measurements (LSM) were also found closely related to
fibrosis stage (Kettaneh et al.,2007).
TE allows the assessment of liver fibrosis by calculating the velocity of a lowfrequency transient shear wave produced by a mechanical probe that is placed directly
on the skin of the patient. The velocity of the wave that penetrates the liver tissue
depends on the stiffness of the liver, which in turn correlates with the extent of liver
fibrosis. In practice, a probe is placed in an intercostal space at a position that is
comparable to the position for standard liver biopsy. Ten successful measurements are
usually necessary for the assessment of liver stiffness. This can be done in less than 5
minutes. This method is easy to learn and quick, results are available immediately,
and a technical assistant can perform the procedure (Sandrin et al.,1999).
Comparison of elastic ratio with various markers and scores showed that elastography
appears to be a better technique than Forns score, hepascore, fibro index and serum
markers like hyaluronic acid (HA) (Yohei et al., 2011).
Doppler analysis:
Doppler measures the velocity of blood flow hemodynamic variations in hepatic
vasculature, as sever fibrosis causes irregularities and abnormalities in hepatic blood
vessels. Recent data indicate a close correlation between arterioportal ratio and degree
]40[
of fibrosis, higher ratio indicates severe fibrosis (F3-F4) and low ratio shows
moderate fibrosis (F1-F2) (Abu-Yousef 1991;Albrecht et al.,1999;Hirata et al.,
2001;Bonekamp et al.,2009).
Magnetic resonance imaging (MRI):
MRI observes changes in hepatic parenchyma. Noninvasive prognosis of liver
cirrhosis is proposed by using double contrast material-enhanced MR imaging. This
can detect cirrhosis with great sensitivity and specificity of 90%. Combining Doppler
ultrasonography with MRI can give a good picture of liver fibrosis in patients
suffering with chronic HCV (Lucidarme et al.,2003;Numminen et al.,2005;Aguirre
etal.,2006).
Computed tomography (CT):
CT identifies microvascular permeability changes in a model of liver fibrosis. In a
latest study, the severity of liver fibrosis was predicted by heterogeneous
enhancement of the liver; hepatic parameters. Perfusion calculated with a dynamic
contrast-enhanced single-section CT, linked with the severity of chronic liver disease.
However, no well characterized study has specifically evaluated the worth of CT in
diagnosing degree of fibrosis. Therefore, currently its role in diagnosis of liver
fibrosis is still lacking (Taura et al., 2001; Materne et al.,2002 ;Wang et al., 2007).
Modified imaging techniques:

Imaging techniques with modification like Real-time elastography, Tissue strain
imaging, Supersonic shear imaging, Contrast enhanced MRI, Diffusion-weighted
MRI, Magnetic resonance spectroscopy, Positron emission tomography (PET), Single
photon emission computed tomography (SPECT) are also in use to evaluate liver
fibrosis and cirrhosis with considerable limitations like, lack of data and expertise,
high cost, radiation exposure and short half-life of the tracer in PET and
SPECT(Ahmad et al.,2011).
]41[
Combined Imaging and markers:

Fibroscan + Fibrotest:
The combination of two useful noninvasive methods, fibroscan and fibrotest have
been validated as non-invasive markers of METAVIR fibrosis stages from F0 to F4
using biopsy, and as prognostic markers of liver related mortality in patients with
CHC (Poynard et al.,2014 ; Shaheen and Myers 2008 ;Castera and 2005).
In summary, surrogate markers may support the clinical decision making process, but
a single surrogate marker or score cannot replace the liver biopsy. On the other hand,
attempts have been made to combine different surrogate markers and biopsy in
clinical decision algorithms that aim to reduce the need for liver punctures. Increasing
efforts are being made to combine surrogate markers with transient elastography, and
it seems that some progress can be expected in precise prediction of different fibrosis
stages, which may eventually replace liver biopsy for fibrosis staging. But till now
Liver biopsy remains an imperfect golden standard. Imaging techniques, serum
biomarkers and biomarker panels are advancing along the route to the clinic stage, but
require extensive validation (Baranova et al.,2011).
ANTIFIBROTIC THERAPIES:
Liver fibrogenesis was traditionally considered a passive and irreversible process,
due to the collapse of the hepatic parenchyma and its substitution by a collagen-rich
tissue (Al-harbi et al., 2012).
However, a number of intensive clinical and experimental observations during the
past 20 years indicate that it is a dynamic process of tissue repair that develops after
repeated liver injury . The onset of liver fibrosis is usually insidious, and most of the
related morbidity and mortality occur after developing cirrhosis . In most patients,
progression to cirrhosis takes 15-20 years (Leija et al., 2001).
]42[
When fibrosis is not controlled, it can further progress into cirrhosis. In contrast with
the traditional idea that cirrhosis is an irreversible state, there is solid evidence
indicating that fibrosis even cirrhosis could be reversible (Duval et al., 2015).
Dr. Hans Popper, an authority on liver diseases in the USA during the 20th century,
has pointed out that anyone who can stop or delay liver fibrosis would be able to
cure most chronic liver diseases.
Current treatments of cirrhosis include withdrawal of the causative agent and
treatment of complications, but the only curative treatment for advanced cirrhosis is
liver transplantation. Its clinical applicability is, however, limited by shortage of
donors, by the commitment of recipients to life-long toxic immunosuppression, and
by poor condition of the potential recipient.
Since the progress of fibrosis is basically similar in all liver diseases regardless of the
etiology, the development of antifibrotic therapies should benefit all patients with
fibrosing liver injury. The liver's ability to regenerate and its first-pass metabolism
allow lower doses of orally administered compounds to achieve a therapeutic
response, minimizing systemic distribution and non-hepatic side effects, which makes
liver fibrosis an attractive target for therapy. The progress in clinical research and
basic science and improved diagnostic tools promise to halt the progression of liver
fibrosis and/or promote its reversion. Potential therapeutic approaches for liver
fibrosis include: treatment of the primary disease, suppression of hepatic
inflammation, inhibition of HSCs activation, promotion of matrix degradation,
stimulation of HSCs apoptosis, and targeted antifibrotic therapies). These approaches
will be addressed individually.
Treatment of the primary disease:
]43[
Clearing the primary cause of liver disease is still the most effective intervention in
the treatment of liver fibrosis. Removal of excess iron or copper in haemochromatosis
or Wilsons disease, respectively, abstinence in alcoholic liver disease, antihelminthic
therapy in parasitic infections, clearance of HBV or HCV in chronic viral hepatitis,
and biliary decompression in bile duct obstruction, or, most recently, weight loss in
patients with NASH, lead to reduced fibrogenesis. This approach can be highly
effective when applied early, resulting in a near-normal restitution of hepatic
histology (Dixon et al.,2004 ;Falize et al.,2006 ;Hammel et al.,2001 ;Klein et
al.,2006 ;Roberts et al.,2007).
Suppression of hepatic inflammation:
In sustained liver injury, persistent inflammation perpetuates the fibrogenic HSCs
phenotype and leads to fibrosis. Therefore, a number of anti-inflammatory agents
have been evaluated in vitro and in vivo. Treatment with corticosteroids in patients
with severe autoimmune hepatitis leads to hepatic fibrosis only in a minority of
patients (Czaja and Carpenter 2004). Blocking interleukin 1 by expressing the
interleukin-1 receptor antagonist, also reduced liver damage and pro-inflammatory
cytokine levels in an in vivo model of ischaemia-reperfusion injury) (Harada et
al.,2002).
Recombinant interleukin 10 reduces inflammation and fibrosis in patients with
chronic hepatitis C, but results in an increased viral load. (Nelson et al.,2003). Local
expression
of
interferon
alpha
(IFN)
improved
liver
fibrosis
in
dimethylnitrosamine-induced model of cirrhosis(Suzuki et al.,2003).

Finally, the beneficial effects of ursodeoxycholic acid in the treatment of primary
biliary cirrhosis (PBC) are thought to be in part via anti-inflammatory mechanisms
(Pares et al.,2006).
Inhibition of HSCs activation:
]44[
HSCs activation, proliferation and fibrogenesis are pivotal steps in the development
of liver fibrosis, making the mechanisms that mediate them attractive targets for
therapeutic intervention. Oxidative stress, which stimulates HSCs activation, is one
such target. Several therapeutic agents have been tested in animal models to
counteract the activation of HSCs by oxidative stress, like vitamin E and C (Soylu et
al.,2006), and S-adenosyl- L- methionine (Yang et al.,2009). but the effects were
minor. A number of other agents have been shown to inhibit the HSC activation. They
include interferon gamma (IFN) (Li et al.,2012).
and ligands for peroxisomal
proliferator activated receptor (PPAR) gamma (Rockey 2008).

TGF antagonists are extensively tested, since neutralizing of this potent cytokine
would have the dual effect: inhibition of matrix production and acceleration of its
degradation. Adenoviral expression of Tgf1 antisense mRNA, and gene transfer of
Smad7, which blocks TGF intracellular signaling, also had positive effect. Gene
transfer of soluble TGFR2 thatinhibits liver fibrogenesis in rats is shown to attenuate
apoptosis, injury, and fibrosis in bleomycininduced lung fibrosis in mice. Blocking
TGF activity by synthetic peptide reduces fibrosis in mice in carbon tetrachloride
(CCL4)-induced liver fibrosis, and bleomycin-induced skin and lung fibrosis
(Hayashi and Sakai 2012 ;Hawinkels and Ten2011).Although inhibitors of TGF1
are effective in short-term animal models, they are not suitable for long term therapy
because of the significant role of TGF1 in homeostasis and repair(Kisseleva and
Brenner 2006).
Promotion of matrix degradation:
Remodeling of ECM occurs through the action of MMPs that efficiently cleave
collagens and other matrix components, and the TIMPs, critical determinants of
fibrosis reversal, which inhibit their activity. During the regression, decreased TIMP1 levels are associated with clearance of activated HSCs through apoptosis
(Hemmann et al.,2007).
]45[
Fibrinolysis and matrix rearrangement can be induced by a number of agents, as
shown by studies in animal models of liver fibrosis. For example, direct adenoviral
administration of Mmp1 mRNA attenuated established liver fibrosis in thioacetamide
treated rats, while administration of an anti-TIMP1 antibody decreased HSCs
activation and attenuated fibrosis in a CCl4 rat model (Iimuro et al.,2003 ;Parsons et
al., 2004).
Downregulation of TIMPs by the reproductive hormone relaxin also inhibits liver
fibrosis in vitro(Williams et al.,2001).Furthermore, adenoviral delivery of urokinasetype plasminogen activator (uPA), which initiates the matrix proteolysis and
upregulates hepatocyte growth factor (HGF))(Narmada et al.,2012).
results in enhanced collagenase activity, fibrosis regression and hepatocyte
regeneration (Salgado et al.,2000). MMPs gene delivery in advanced liver fibrosis
shows that even transient shifts in the imbalance between MMPs and TIMPs are
sufficient for the resolution - if the underlying causative stimuli have been
successfully removed.(Iimuro and Brenner 2008).
Dietary supplementation with zinc, an essential co-factor for MMP activity, has also
been shown to promote collagen degradation in CCl4 injured rats (Gimenez et
al.,1994).
Collagen synthesis is also sensitive to changes in food intake, and malnutrition may
have profound effects on its production(Spanheimer et al.,1991).
Stimulation of HSCs apoptosis:
Apoptosis is a key event in the spontaneous recovery from liver fibrosis (Iredale et
al.,1998). and can be induced by two general mechanisms(Canbay et al.,2004).
The first, extrinsic pathway, works via stimulation of specific cell surface death
receptors, which trigger an intracellular cascade of caspases, ensuing cellular
apoptosis. Second, intrinsic pathway, operates at the level of the mitochondria.
Stimuli like toxins, UV radiation, and reactive oxygen species stimulate the
]46[
proapoptotic factors in the mitochondrial membrane, resulting in leakage of
cytochrome C into the cytosol, caspase activation, and apoptosis. In addition, upon
activation of HSCs, the expression of a cell surface death receptors CD95 (APO1/Fas) and CD95L (APO-1-/Fas-ligand) increases, which may explain the increased
apoptosis seen upon their activation in vitro and in vivo (Saile et al.,1997).
Also, activation of TNF receptor, and low-affinity nerve growth factor (NGF)
receptor (p75) increases HSCs apoptosis in vitro (Trim et al.,2000).Transformed
HSCs also express peripheral benzodiazepine receptors, which render the cells
sensitive to peripheral benzodiazepine receptor-ligand-induced apoptosis (Fischer et
al.,2001). NFB, on the other hand, protects HSCs from apoptosis, and its inhibition
with gliotoxin accelerates recovery from drug-induced liver fibrosis in rats(Wright et
al., 2001), proving that HSCs pro-apoptotic agents could abrogate liver fibrosis. The
approaches mentioned in this section may sound attractive for in vitro work, but the
selectivity may be too low to use this approach in vivo.
Targeted antifibrotic therapy:

Wound healing is an important universal process, hence liver-specific targeting of
antifibrotic therapies minimizes the potential harmful effects on other tissues.
Furthermore the extensive first pass metabolism makes liver an attractive target for
directed therapy with orally administered drugs. Still, hepatic delivery needs to be
optimized via cell-specific targeting (of hepatocytes, Kupffer cells, or HSCs). It has
been shown, for instance, that administration of recombinant insulin-like growth
factor 1 (rIGF1) or HSCs-targeted induction of its expression using a viral vector in
CCl4-treated mice and rats, significantly reduced liver fibrosis (Castilla-Cortazar et
al.,1997; Sanz et al.,2005 ;Sobrevals et al., 2010).
Treatment of cirrhotic rats with rIGF1 promotes weight gain, nitrogen retention, and
intestinal absorption of nutrients and was able to exert hepatoprotective effect
(Castilla-Cortazar et al., 1997),Concerns regarding the safety of gene therapy with
]47[
regard to genotoxicity and effects on the immune system remain. However, with
improved delivery techniques and the conditional expression systems, the antifibrotic
trials in humans are no longer a matter of (distant) future.
]48[

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Review of Literature

araising from the stomach, intestine and spleen

scarring process is a common feature of most chronic diseases. In epithelial organs,

Causes of chronic liver disease:

accumulate. Any chronic perturbation of hepatic homeostasis, whether visible by light

Biological Principals of Hepatic Fibrosis:

4-Soluble factors (cytokines / peptides) and cell-matrix interactions are

2012).Potential reversibility of hepatic fibrosis varies depending on the duration of

In response to liver injury, HSC undergo rapid activation, functional and

Activation consists of two major phases: initiation and perpetuation, followed by

contractility,matrix degradation,retinoid loss,and WBC chemoattractant/cytokine

Liver fibrosis scoring/staging

Importance of liver fibrosis staging:

(Myers et al.,2008).The most frequent

Classification of Liver Histology

Procollagen type III amino-terminal peptide (PIIINP):

concentration in the basement membrane is higher in hepatic fibrogenesis (Veidal et

Matrix metalloproteinases and their inhibitors:

Combined direct markers:

Serum ALT, AST and AFP levels:

Combined indirect markers:

Fibro-quotient (FibroQ) index:

Composite fibrosis markers:

Transient Elastography (FibroScan):

Modified imaging techniques:

Combined Imaging and markers:

dimethylnitrosamine-induced model of cirrhosis(Suzuki et al.,2003).

and ligands for peroxisomal

proliferator activated receptor (PPAR) gamma (Rockey 2008).

Targeted antifibrotic therapy:

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