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The liver is a central organ in the human body, coordinating several key metabolic roles. The structure of the liver which consists of the distinctive arrangement of hepatocytes, hepatic sinusoids, the hepatic artery, portal vein and the central vein, is critical for its function. Due to its unique position in the human body, the liver interacts with components of circulation targeted for the rest of the body and in the process, it is exposed to a vast array of external agents such as dietary metabolites and compounds absorbed through the intestine, including alcohol and drugs, as well as pathogens. Some of these agents may result in injury to the cellular components of liver leading to the activation of the natural wound healing response of the body or fibrogenesis. Long-term injury to liver cells and consistent activation of the fibrogenic response can lead to liver fibrosis such as that seen in chronic alcoholics or clinically obese individuals. Unidentified fibrosis can evolve into more severe consequences over a period of time such as cirrhosis and hepatocellular carcinoma. It is well recognized now that in addition to external agents, genetic predisposition also plays a role in the development of liver fibrosis. An improved understanding of the cellular pathways of fibrosis can illuminate our understanding of this process, and uncover potential therapeutic targets. Here we summarized recent aspects in the understanding of relevant pathways, cellular and molecular drivers of hepatic fibrosis and discuss how this knowledge impact the therapy of respective disease.
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Cellular Mechanisms of Liver Fibrosis
Pragyan Acharya
1
*, Komal Chouhan
1
, Sabine Weiskirchen
2
and Ralf Weiskirchen
2
*
1
Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, India,
2
Institute of Molecular Pathobiochemistry,
Experimental Gene Therapy and Clinical Chemistry, RWTH University Hospital Aachen, Aachen, Germany
The liver is a central organ in the human body, coordinating several key metabolic roles.
The structure of the liver which consists of the distinctive arrangement of hepatocytes,
hepatic sinusoids, the hepatic artery, portal vein and the central vein, is critical for its
function. Due to its unique position in the human body, the liver interacts with components
of circulation targeted for the rest of the body and in the process, it is exposed to a vast
array of external agents such as dietary metabolites and compounds absorbed through
the intestine, including alcohol and drugs, as well as pathogens. Some of these agents may
result in injury to the cellular components of liver leading to the activation of the natural
wound healing response of the body or brogenesis. Long-term injury to liver cells and
consistent activation of the brogenic response can lead to liver brosis such as that seen
in chronic alcoholics or clinically obese individuals. Unidentied brosis can evolve into
more severe consequences over a period of time such as cirrhosis and hepatocellular
carcinoma. It is well recognized now that in addition to external agents, genetic
predisposition also plays a role in the development of liver brosis. An improved
understanding of the cellular pathways of brosis can illuminate our understanding of
this process, and uncover potential therapeutic targets. Here we summarized recent
aspects in the understanding of relevant pathways, cellular and molecular drivers of
hepatic brosis and discuss how this knowledge impact the therapy of respective disease.
Keywords: liver brosis, cytokines, chemokines, NASH, therapy, alcohol, cholestasis, drugs
INTRODUCTION
The liver is the largest solid organ in the human body, weighing about 1,2001,500 g, and comprising
about 1/50th of the total body weight in an adult (Dooley et al., 2018). Understanding the complex
architecture of the liver is key to understanding liver brosis and its consequences.
The liver has two major sources of blood supply, namely (i) the portal vein and (ii) the hepatic
artery. The portal vein brings venous blood from the intestines and spleen to the liver. The hepatic
artery brings arterial blood to the liver from the celiac axis. The liver is encapsulated by the Glissons
capsule which is mainly composed of connective tissue (Junqueira and Carneiro, 2002). Within the
Glissons capsule, the liver is divided into polygonal sections called lobules which are also separated
by connective tissue. Each lobule has a characteristic arrangement which is disturbed during liver
brosis and is completely damaged during cirrhosis (Figure 1A)(Sasse et al., 1992). Since liver
function is so intricately linked to this arrangement, hepatic function is completely disrupted during
cirrhosis leading to complications. The liver lobule, which is roughly hexagonal, harbors the hepatic
central vein at its center (Sasse et al., 1992;Junquiera and Carneiro 2002). Hepatocytes are the most
abundant cell type in the liver, constituting about 60% of the total cell number and 80% of liver cell
volume. Hepatocytes perform the major roles of the liver such as detoxication of xenobiotics, urea
cycle and the synthesis of plasma proteins (Zhou et al., 2016). Hepatocytes are arranged in straight
Edited by:
Leo A. van Grunsven,
Vrije University Brussel, Belgium
Reviewed by:
Sonia Michael Najjar,
Ohio University, United States
Stefaan Verhulst,
Vrije University Brussel, Belgium
*Correspondence:
Pragyan Acharya
pragyan.acharya@aiims.edu
Ralf Weiskirchen
rweiskirchen@ukaachen.de
Specialty section:
This article was submitted to
Gastrointestinal and Hepatic
Pharmacology,
a section of the journal
Frontiers in Pharmacology
Received: 24 February 2021
Accepted: 21 April 2021
Published: 06 May 2021
Citation:
Acharya P, Chouhan K, Weiskirchen S
and Weiskirchen R (2021) Cellular
Mechanisms of Liver Fibrosis.
Front. Pharmacol. 12:671640.
doi: 10.3389/fphar.2021.671640
Frontiers in Pharmacology | www.frontiersin.org May 2021 | Volume 12 | Article 6716401
REVIEW
published: 06 May 2021
doi: 10.3389/fphar.2021.671640
lines radiating out from the central vein toward the edge of the
lobule. The space between the radially arranged les of
hepatocytes is commonly termed the sinusoids. Bile duct,
lymphatics, neurons, as well as the branches of hepatic artery
and portal vein line the periphery of the lobules and feed into the
liver sinusoids. The portal vein and hepatic artery branch into the
liver sinusoids, toward the central vein. Sinusoids are lined with
fenestrated endothelial cells, and harbor immune cells such as
Kupffer cells, hepatic stellate cells (HSCs) and hepatic natural
killer cells (NK cells). These are known as the non-parenchymal
cells of the liver. The space between the periphery of the
hepatocyte lining and the endothelial cells is known as the
space of Disse. The space of Disse is where the exchange of
nutrients and other molecules occurs between the hepatocytes
and blood owing through the blood capillaries from the portal
vein and the hepatic artery (Sasse et al., 1992). Interactions
between the parenchymal and non-parenchymal cells in this
carefully preserved architecture are central to efcient
functioning of the liver.
Fibrogenesis is a normal wound healing response to tissue
injury. All hepatocellular injuries activate the brogenic
pathways. Once these pathways are activated, brogenic
components of the extracellular matrix (ECM) are secreted
into the space of Disse in order to encapsulate and isolate the
damaged portion of the tissue for repair (Bataller and Brenner,
2005). During the encapsulation, there is an inltration of
immune cells that clear cellular debris and initiate tissue
repair. The transition from a normal liver to brotic liver
involves activation and modulation of complex signaling
pathways, cell-cell communication between the hepatocytes
and non-parenchymal cells, immune system, tissue repair
pathways and the extracellular space. In the normal liver, the
ECM present in the space of Disse is made up of glycoproteins
like bronectin and laminin, type IV collagen (non-brogenic)
and proteoglycans such as heparan sulfate (Figure 1A). These
components form a lattice-like matrix, which are essential for
providing both mechanical support as well as molecular signals
for the proper arrangement and functioning of liver cells. When
there is hepatic injury, the composition and density of the ECM
changes. There is almost a 68 fold increase in the production of
ECM components. Non-brogenic type IV collagen is replaced by
brogenic type I and II collagen (Figure 1B). There is additional
secretion of bronectin, hyaluronic acid and α-smooth muscle
actin into the ECM. In addition, endothelial cell fenestrations as
well as microvilli on the hepatocyte basal membrane are lost
thereby compromising exchange of nutrients and metabolites as
well as other signaling molecules between the circulation and
hepatocytes. While the response to tissue injury is a rapid process
and brogenesis is intended at promoting wound healing,
repeated injury and activation of the brogenic pathways
result in a chronic activation of brogenesis (Iredale et al.,
2013). This leads to an increased synthesis and decreased
degradation of type I collagen over a period of time. This
results in deposition of type I collagen in the ECM
FIGURE 1 | Liver architecture in healthy liver and brosis. (A) In normal liver, hepatocytes are arranged in rows radiating outwards from the central vein, toward the
edge of the lobule. The gaps between the hepatocyte rows are known as sinusoids which are lined with endothelial cells, and contain Kupffer cells, hepatic stellate cells,
and contain extracellular material such as the non brogenic type IV collagen. Hepatic portal vein, hepatic artery and biliary tree are the three major vessels feeding into the
sinusoids and the exchange of blood gases, nutrients and other signaling molecules occurs in the sinusoids. (B) Injury to hepatocytes due to any of several causes
such as alcohol, drug, genetic predisposition, etc., activates the wound healing brogenic response. Chronic injury to the hepatocytes and chronic activation of the
brogenic pathway in the liver leads to synthesis of brogenic type I collagen by the Hepatic stellate cells and its deposition within the sinusoids. Deposition around the
central vein and around the portal vein leads to increase in vascular resistance and portal hypertension. Compensatory mechanisms such as esophageal varices and
ascites follow.
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Acharya et al. Mechanisms of Liver Fibrosis
TABLE 1 | Genetic causes predisposing the liver to brosis.
Disease Gene Gene function Cause
of tissue injury
Clinical presentation with
liver involvement
Wilsons Disease ATP7B Copper transport Intra-hepatic Cu
2+
accumulation Variable presentation. Can be
asymptomatic or accompanied by
brosis, acute hepatitis, end stage liver
disease
Progressive familial
intrahepatic cholestasis
type 3
ABCB4 Biliary phospholipid secretion Accumulation of phospholipids and other
xenobiotics; impairment of bile formation
Manifests in early childhood, jaundice,
splenomegaly, portal hypertension and
physical and mental retardation
Hereditary fructose
intolerance
ALDOB Converts fructose into trioses for entry
into glycolysis and gluconeogenesis
Accumulation of fructose 1 phosphate
and depletion of inorganic phosphate
levels, inhibition of glycogenolysis,
accumulation of high levels of fructose
can be hepatotoxic
Hereditary fructose intolerance,
hepatotoxicity, liver dysfunction
progressing to cirrhosis
Glycogen storage
disease type IV
GBE1 Glycogen branching enzyme Accumulation of unbranched glycogen
causing hepatotoxicity
Variable presentation. Hepatic classical
presentation includes liver dysfunction
progressing to cirrhosis, failure to thrive
by 5 years of age. The non-progressive
hepatic subtype present with
hepatomegaly, liver dysfunction,
myopathy, and hypotonia; but likely to
survive without further progression to
cirrhosis
Tyrosinemia type I FAH Last step in tyrosine catabolism Accumulation of fumarylacetoacetate and
tyrosine in the hepatocytes and oxidative
damage to cells
Presentation as liver or renal failure; in
early infancy; liver related symptoms are
hypoalbunimea, lowering of synthetic
functions of the liver, leading to
steatosis, cirrhosis and HCC
Hemochromatosis HFE Interactions with the transferrin
receptor and iron uptake
Intra-hepatic iron overload Presentation as liver cirrhosis
Argininosuccinate lyase
deciency
ASL Urea cycle enzyme that cleaves
argininosuccinate into arginine and
succinate
Accumulation of urea cycle intermediates,
especially ammonia
Two forms:-Early onset in infancy
associated with hyperammonimea and
vomiting, failure to thrive, or late onset
associated with hyperammonimea
episodes, cirrhosis and neurological
symptoms
Citrin deciency SLC25A13 Calcium binding mitochondrial carrier
protein Aralar2 (exchange of
cytoplasmic glutamate with
mitochondrial aspartate across the
inner mitochondrial membrane)
Citrullinemia and ammonia accumulation Neonatal intrahepatic cholestasis:
impaired bile ow, brosis, cirrhosis; late
onset citrullinemia 2: neuropsychiatric
symptoms
Cholesteryl ester storage
disease
LIPA Lysosomal acid lipase (LAL) catalyses
the intracellular hydrolysis of
triacylglycerols and cholesteryl ester
Intracellular accumulation of cholesteryl
esters, triglycerides in the lysosomal
compartment of hepatocytes
Early onset: hepatomegaly,
splenomegaly and altered serum
transaminases
α1 antitrypsin deciency SERPINA1 Inhibitor of various proteases including
trypsin and therefore, protects cells
from inammatory proteases such as
from neutrophils
Accumulation of mutant poly-AAT bers
leading to hepatotoxicity
Variable clinical severity ranging from
chronic hepatitis and cirrhosis to
fulminant liver failure
Cystic brosis CFTR Membrane chloride channel;
expressed on the cholangiocytes
Pathogenesis unknown Age of onset is late: elevation of serum
liver enzymes, hepatic steatosis, focal
biliary cirrhosis, multilobular biliary
cirrhosis, neonatal cholestasis,
cholelithiasis, cholecystitis and micro-
gallbladder
Alström syndrome ALMS1 Centrosome and basal body
associated protein: microtubule
organization
Pathogenesis unknown: likely to be
involved in cellular Ca
2+
signaling
Multiple organ dysfunction: liver
involvement can range from
steatohepatitis to portal hypertension
and cirrhosis and can cause hepatic
encephalopathy and life-threatening
esophageal varices
(Continued on following page)
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Acharya et al. Mechanisms of Liver Fibrosis
surrounding the lobules which is the hallmark of brosis. Large
amounts of type I collagen deposition around the lobules and
within the sinusoidal space disrupts the radial arrangement of the
hepatocytes, interfering in the ow of nutrients and signaling
molecules from the blood through the sinusoids to the
hepatocytes, disrupting hepatocyte function. The deposition of
collagen around the lobules causes major structural changes in
the liver thereby disrupting liver function as well (Iwakiri, 2014).
Accumulation of type I collagen bers leads to mechanical
rigidity in the ECM which puts pressure on the blood vessels
owing through the liver. This leads to intrahepatic
vasoconstriction and vascular resistance (Pinzani and Vizzuti,
2005). Therefore, in a major vein like the hepatic portal vein, this
leads to portal hypertension, which is a major clinical concern in
liver brosis. The unchecked development of portal hypertension
has two major consequences: (i) development of collateral blood
vessels from the systemic and splanchnic circulation and, (ii)
vasodilation of the hepatic artery (Bosch, 2007). This further
increases blood ow into the hepatic portal vein leading to further
portal hypertension. As a compensatory mechanism to relieve
pressure, submucosal veins present at the lower portion of the
esophagus dilate leading to esophageal varices that have the
potential to rupture and can be fatal. Fluid begins
accumulating within the peritoneal cavity, leading to ascites-
the hallmark of advanced decompensated cirrhosis (Bolognesi,
et al., 2014;Perri, 2013). These physiological processes are
summarized in Figure 1B.
DRIVERS OF LIVER FIBROSIS
Genetic Disorders
Several genetic diseases predispose the liver to brosis (Scorza
et al., 2014). In all of these diseases, the initiation of brosis begins
with tissue injury due to a consequence of the genetic defect
followed by a brogenic wound healing response, as discussed
above. Genetic causes for liver brosis have come into light due to
the advancements in molecular genetic and imaging techniques.
Several genetic polymorphisms summarized in Table 1, have
been implicated in the occurrence of liver brosis, leading to
cirrhosis (Pinzani and Vizzutti, 2005). Most of these mutations
affect many different cell types but predispose the individual to
liver brosis and in some cases, liver cirrhosis (Scorza et al., 2014).
Many of the genes listed in Table 1, such as, ABCB4,ALDOB,
GBE1,FAH,ASL,SLC25A13, and SERPINA1 are highly expressed
in the liver and therefore, mutations in these genes, the liver is the
organ which is most affected. Most genetic disorders that lead to
cirrhosis manifest in childhood and are a leading cause of
pediatric liver cirrhosis, apart from childhood obesity (Pinto
et al., 2015). In addition to the genetic mutations that
predispose individuals to hepatic brosis that appear in
childhood, mutations of the PNPLA3 gene have been
described as a major predisposing factor in non-alcoholic fatty
liver disease (NAFLD) (Anstee et al., 2020). PNPLA3 encodes for
Patatin-like phospholipase domain-containing protein 3 or
adiponutrin and is abundantly expressed in hepatocytes,
adipocytes as well as HSCs (Dong, 2019). The PNPLA3 I148M
variant has been shown to have a positive association with hepatic
fat content (steatosis), NAFLD, non-alcoholic steatohepatitis
(NASH) as well as hepatocellular carcinoma (Dong, 2019).
The global prevalence of NAFLD is about 25% and in obese
individuals or in the presence of type 2 diabetes mellitus, it
increases to about 60% (Younossi et al., 2016). Therefore,
PNPLA3 gene is a strong predisposing genetic factor for
hepatic brosis. Although the PNPLA3 protein has been
shown to have triacylglycerol lipase and acylglycerol
transacylase enzymatic activities, its exact role in hepatocytes
have been controversial (Jenkins et al., 2004;Dong, 2019). Other
studies have demonstrated a retinyl esterase activity for PNPLA3
(Pirazzi et al., 2014). HSCs are reservoirs for retinoic acid, which
activate the retinoic acid receptor (RAR) mediated transcription
which keeps brogenesis under control (Hellemans, et al., 1999;
Hellemans et al., 2004;Wang et al., 2002). Mutations in the
PNPLA3 gene that alter the retinyl esterase activity therefore,
decrease the level of retinoic acid in the HSCs and therefore
reduce the RAR mediated control of brogenesis in HSCs
(Bruschi et al., 2017). However, it is now recognized that
PNPLA3 has pleiotropic roles in the hepatocyte that are still
TABLE 1 | (Continued) Genetic causes predisposing the liver to brosis.
Disease Gene Gene function Cause
of tissue injury
Clinical presentation with
liver involvement
Congenital hepatic
brosis
Cryptogenic
causes
NA NA Multiple organ brosis and dysfunction:
Can present as the following in case of
liver involvement: (i) portal hypertension
(most common and more severe in the
presence of portal vein abnormality), (ii)
cholangitis with cholestasis and
recurrent cholangitis, (iii) both portal
hypertension and cholangitic
symptoms; and (iv) latency that appears
at a late age with hard hepatomegaly
Non-alcoholic fatty liver
disease (NAFLD)
PNPLA3 Pleiotropic role with triglyceride lipase
and retinyl esterase activity
Accumulation of triglycerides, impaired
retinoic acid receptor signaling and
activation of HSC brogenic pathway
Hepatic steatosis, brosis, cirrhosis,
hepatocellular carcinoma
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Acharya et al. Mechanisms of Liver Fibrosis
under investigation such as in hepatocyte lipid droplet
homeostasis, HSC quiescence and proliferation regulation
(Dong, 2019). As several roles for PNPLA3 are suggested,
PNPLA3 might be a good therapeutic target to control
NAFLD related brosis and disease progression.
Alcohol
Excessive and continued alcohol intake over large periods of
time, i.e., alcohol abuse, can lead to liver brosis followed by
cirrhosis and liver cancer (Stickel et al., 2017). Alcoholic liver
disease (ALD) comprises a spectrum of liver disorders ranging
from fatty liver, steatosis, brosis with varying degrees of
inammation, cirrhosis. Alcohol abuse contributes to almost
50% of chronic liver disease related deaths globally (Rehm and
Shield 2019). While the pathophysiology of alcohol induced
cirrhosis is not completely understood, alcohol and its
metabolic intermediates such as acetaldehyde are thought to
play an important role in it. Alcohol is absorbed from the
duodenum and upper jejunum by simple diffusion, reaching
peak blood concentration by 20 min post ingestion after which it
is quickly redistributed in vascular organs (Koob et al., 2014).
Alcohol cannot be stored and needs to undergo obligatory
oxidation which occurs predominantly in the liver (Figure 2)
(Yang et al., 2019). The rst step in alcohol oxidation converts
alcohol into acetaldehyde. There are three enzymes in the liver
that can carry out this reaction (i) alcohol dehydrogenase
(ADH) which catalyzes the bulk of ethanol to acetaldehyde
conversion, (ii) the alcohol inducible liver cytochrome P450
CYP2E1 (microsomal ethanol oxidizing system or MEOS) and,
(iii) peroxisomal catalase. The ethanol to acetaldehyde
conversion by ADH generates NADH (Berg et al., 2002).
Oxidation of large amounts of alcohol therefore, leads to the
accumulation of NADH, which inhibits lactate to pyruvate
conversion and promotes the reverse reaction. Lactate to
pyruvate conversion is an important means of entry of
lactate into gluconeogenesis. As a result, lactic acidosis and
hypoglycemia may occur during excessive alcohol consumption.
NADH/NAD
+
ratio also allosterically regulates fatty acid
β-oxidation which breaks down long chain acyl CoA to
acetyl CoA for entry into TCA cycle (Berg et al., 2002). Since
NADH is a product of fatty acid oxidation, an increase in
NADH/NAD
+
ratio provides an allosteric feedback to the
fatty acid β-oxidation pathway thereby decreasing the
catabolism of fatty acids and leading to their intracellular
accumulation. This leads to fatty liver.NADH also inhibits
two enzymes of the TCA cycle-isocitrate dehydrogenase and
α-ketoglutarate dehydrogenase thereby decreasing the
consumption of acetyl CoA by the TCA cycle and leading to
increase in intra-hepatic acetyl CoA levels. The accumulation of
acetyl CoA, in turn, leads to the increased production and
release of ketone bodies exacerbating the acidosis already
present in the blood due to increased levels of lactate
(McGuire et al., 2006). This is known as alcoholic
ketoacidosis, which creates a medical emergency. At very
high levels of ethanol consumption, the metabolism of
acetate becomes compromised leading to the accumulation of
acetaldehyde within the hepatocytes. Acetaldehyde can modify
the functional groups of many proteins and enzymes
irreversibly forming acetaldehyde adducts which leads to a
global dysfunction of hepatocytes and eventually, to cell
death (Setshedi et al., 2010). The second major pathway for
ethanol metabolism is via the inducible cytochrome P450
CYP1E2, also known as the microsomal ethanol oxidizing
system (MEOS) (Lieber, 2004). This is located in the smooth
FIGURE 2 | Alcohol metabolism in the liver. Three pathways are involved in alcohol metabolism and all of them converge on the oxidation of ethanol to
acetaldehyde. Acetaldehyde is further converted to acetate by aldehyde dehydrogenase in the mitochondria. Acetate can be rapidly oxidized into CO
2
and H
2
Oby
peripheral tissues, or can be diverted to the tri-carboxylic acid (TCA) pathway. The oxidation of ethanol to acetal dehyde by microsomal ethanol oxidation system (MEOS)
occurs in the smooth endoplasmic reticulum and changes the NADPH/NADP ratio which in turn inuences the regeneration of glutathione thereby increasing
cellular oxidative stress. The alcohol dehydrogenase pathway is the major pathway and occurs in the cytosol, generating large amounts of NADH.NADH in turn inhibits
TCA cycle enzymes and leads to accumulation of acetyl CoA and increase in ketone body generation and acidosis. NADH also inhibits fatty acid oxidation leading to
accumulation of fats and causing fatty liver.A combination of the above factors leads to tissue injury and activation of the brogenic pathway.
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Acharya et al. Mechanisms of Liver Fibrosis
endoplasmic reticulum of hepatocytes. In normal people with
average to below average alcohol consumption, MEOS forms a
minor pathway for intracellular alcohol metabolism (Lieber,
2004). However, it increases manifold upon chronic alcohol
consumption. MEOS catalyzes a redox reaction converting
molecular oxygen to water and NADPH to NADP
(Figure 2). In the liver, glutathione plays an important role
in maintaining the cellular redox status and participates in
xenobiotic metabolism (Yuan and Kaplowitz, 2009). NADPH
is essential in the regeneration of glutathione. The consumption
of cellular NADPH leads to a decrease in regeneration of
glutathione thereby leading to oxidative stress. This results in
cell death and inammation leading to alcoholic hepatitis
which, in itself can be fatal (Morgan, 2007). Often, these
processes occur hand in hand. Cellular depletion of
glutathione has an additional consequence. Glutathione is
required for the detoxication of several drugs including
acetaminophen (van de Straat et al., 1987). In the
hepatocytes, acetaminophen is modied to form a cytotoxic
metabolite known as N-acetyl-p-benzoquinone imine (NAPQI)
via CYP2E1 (van de Straat et al., 1987). Conjugation of NAPQI
to glutathione results in an S-glutathione product that detoxies
the molecule and allows safe excretion in the urine. However,
depletion of glutathione reserves allows unconjugated NAPQI
to prevail in the cells which reacts with DNA and proteins to
form adducts, thereby causing cytotoxicity and hepatocyte
death (Macherey and Dansette, 2015). Long term alcohol use
induces CYP2E1 and therefore facilitates rapid NAPQI
formationwhentheliverencountersacetaminophen.Atthe
same time, chronic alcohol abuse leads to low glutathione
reserves. A combination of both these changes makes the
liver highly susceptible to acetaminophen induced liver injury
as well as injury due to other drugs or metabolites that go
through the glutathione detoxication pathway. While drug
overuse is, in itself a cause for liver injury, in a background
of alcoholic liver disease, it can lead to massive liver damage.
Damage to hepatocytes, either due to chronic alcohol abuse,
exacerbated by drug use, activates the brogenic pathway
leadingtohepaticbrosis, cirrhosis and hepatocellular
carcinoma.
Drugs
Drugs induce hepatic brosis by causing drug-induced liver
injury (DILI) that causes the initiation of brogenic tissue
repair mechanisms. While the prevalence of DILI is lower as
compared to other causes of liver injury, such as alcohol, hepatisis
or steatosis, it can lead to life-threatening complications. DILI can
be of two types: (i) intrinsic (due to injury caused by a known on-
target drug) or (ii) idosyncratic (due to injury caused by an
unknown factor and cannot be explained by known
pharmacological elements e.g., herbal preparations of
unknown compositions) (DiPaola and Fontana, 2018). Among
intrinsic causes, acetaminophen induced DILI is the most
common. As described above, acetaminophen overload
combined with alcohol abuse can exacerbate the liver injury
that can occur due to either alcohol or acetaminophen alone.
A major function of the liver is detoxication of xenobiotic
compounds that enter our circulation either through diet or
through intravenous drug usage. Detoxication mechanisms in
the liver mainly involve the cytochrome P450 family (CYP gene
families CYP1,CYP2,CYP3)(McDonnell and Dang, 2013)
(Figure 3). Cytochrome P450s are a group of heme proteins
that are involved in the initial detoxication reactions of small
molecules such as dietary and physiological metabolites, as well as
drugs (Zanger and Schwab, 2013;Todorovic Vukotic et al., 2021).
The expression of the CYP genes is inuenced by several factors
such as age, sex, promoter polymorphisms, cytokines, xenobiotic
compounds and hormones, to name a few (Zanger and Schwab,
2013). Cytochrome P450 mainly carry out a monooxygenation
reaction and carry oxidation of drugs/xenobiotic compounds.
FIGURE 3 | Metabolism of drugs and other xenobiotics in the liver. Drug and xenobiotic metabolism occurs in two phases: (i) phase I is catalyzed by the cytochrome
P450 family of monooxygenases which metabolize ingested small molecules to form inert or bioactive metabolic intermediates. (ii) These intermediates are further
catalyzed in phase II reactions to form soluble polar compounds that can be further excreted through urine or bile. Accumulation of bioactive drug or xenobiotic
intermediates can lead to the formation of protein or nucleic acid adducts causing autoimmune reaction, carcinogenesis or direct cellular injury.
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Acharya et al. Mechanisms of Liver Fibrosis
This can either convert the molecule into an inert or bioactive
molecule.
Bioactive compounds can covalently modify intracellular
proteins, leading to direct cellular injury, carcinogenesis or
production of hapten-protein conjugates that can lead to
antibody mediated cytotoxicity (Figure 3). Although the
classical view of DILI is that drugs become hepatotoxic as a
consequence of or defects in their metabolism, several factors
may inuence the nal outcome of drug intake such as age,
gender, comorbidities, intake of alcohol, other drugs or herbal
preparations and polymorphisms of the CYP genes (Tarantino
et al., 2009). The exact mechanism of DILI in speciccases
depends on the nature of the molecule and its CYP-
transformed metabolites. Drug metabolism can generate
free radicals or electrophiles that can be chemically reactive.
This can lead to the depletion of reduced glutathione,
formation of protein, lipid or nucleic acid adducts and lipid
peroxidation. Unless these metabolic intermediates are rapidly
neutralized through phase II reactions, they can contribute to
cellular stress and injury (Figure 3). They can also lead to
modulation of signaling pathways, induce transcription
factors, and alter gene expression proles. In the liver,
accumulation of large quantities of reactive drug
metabolites can lead to hepatocellular injury, formation of
protein adducts that can act as haptens and stimulate
production of auto-antibodies or promote cellular
transformation. Cellular injury then leads to induction of
brogenic responses as described above.
Cholestasis
Cholestasis is emerging as a leading cause for liver injury and
brosis. Cholestatic liver diseases can occur due to primary
biliary cirrhosis and primary sclerosing cholangitis and involve
injury to the intra- and extra-hepatic biliary tree (Penz-
Österreicher et al., 2011). The pathogenesis of cholestasis is
unclear but is believed to have an autoimmune component to it
(Karlsen et al., 2017). I primary sclerosing cholangitis (PSC)
several strictures appear around the bile ducts and cause bile
duct injury. This activates the portal broblasts around the bile
duct, which then differentiate into collagen secreting
myobroblasts (MFBs) similar to those derived from HSC
activation. Recent studies have shown that brogenic MFBs
have inherent heterogeneity and can be derived from both HSCs
and portal broblasts (Karlsen et al., 2017). PSC has been shown
to be associated with a varied manifestation of other diseases
such as inammatory bowel disease, cholangiocarcinoma, high
IgG4 levels, autoimmune hepatitis and colonic neoplasia (Wee
et al., 1985;Broomé et al., 1992;Perdigoto et al., 1992;Siqueira
et al., 2002;Mendes et al., 2006;Berntsen et al., 2015). Due to its
association with autoimmune responses, PSC is thought to
involve a genetic predisposition which is activated by an as
yet unidentied environmental trigger such as gut dysbiosis
(Rossen et al., 2015). Although PSC is traditionally recognized
asararedisease,itsincidenceisontheriseduetoanincreasein
unknown environmental triggers (Karlsen et al., 2017).
Therefore, the pathogenesis of PSC is varied and injury to
the bile ducts can occur through multiple pathways.
However, the resultant bile duct injury leads to activation of
the portal broblasts and consequent brogenesis.
Metabolic Disorders: Non-alcoholic Fatty
Liver Disease and Non-alcoholic
Steatohepatitis
The metabolic syndrome is a group of associated diseases that
increase cardiovascular risk factors and are linked with obesity
and type 2 diabetes mellitus (Rosselli et al., 2014). Liver
manifestations of the metabolic syndrome result in NAFLD
(Rosselli et al., 2014). NAFLD is attaining epidemic
proportions all over the world. The global prevalence of
NAFLD is about 25% and in obese individuals or in the
presence of type 2 diabetes mellitus, it increases to about 60%
(Younossi et al., 2016). NAFLD is linked to increased risk of
hepatic brosis, hepatocellular carcinoma and mortality due to
cardiovascular disease. The more severe subtype of NAFLD is
NASH, which has a global prevalence of about 26% and which is
associated with severe hepatic inammation, brosis leading to
cirrhosis and HCC as well as end stage liver disease (Younossi
et al., 2016;Younossi et al., 2019). Recently reported trends in the
incidence of NAFLD over time suggest that NAFLD will become
the leading cause of end stage liver disease in the decades to come.
Emerging data from India, suggests that the national prevalence
of NAFLD is about 932% in the general population and about
53% in obese individuals (Kalra et al., 2013;Duseja, 2010).
Therefore, NAFLD is a global clinical concern. The molecular
pathogenesis of NAFLD is complex. However, all pathways in
NAFLD converge at the conversion of HSCs into probrogenic
MFBs, through the activation of the TGF-βpathway (Buzzetti
et al., 2016)(Figure 4). TGF-βis a pleiotropic cytokine and is
involved in various cellular processes like cell proliferation,
survival, angiogenesis, differentiation, and the wound healing
response (Mantel and Schmidt-Weber, 2011). TGF-βbinds to the
TGF-βreceptor type II, which in turn phosphorylates TGF-β
receptor type I thereby recruiting and phosphorylating the
intracellular signal transducer proteins belonging to the SMAD
superfamily. The SMAD superfamily is composed of intracellular
signal transducers that specically respond to the TGF-βreceptor
modulation. Phosphorylated SMADs subsequently translocate
into the nucleus and control the expression of the TGF-β
regulated target genes (Mantel and Schmidt-Weber, 2011)
(Figure 4). The activation of HSCs via TGF-βplays a major
role in the advanced NAFLD in both experimental animal
models, as well as in human liver injury (Yang et al., 2014). In
addition to HSC activation, TGF-βsignaling followed by SMAD
phosphorylation is known to cause hepatocyte death driving
progression to NASH (Yang et al., 2017). Hepatocyte death via
TGF-βsignaling is accompanied by generation of reactive oxygen
species as well as lipid accumulation in hepatocytes (Yang et al.,
2017). Activation of the TGF-βpathway also leads to HSC
differentiation into MFBs leading to formation of brillar
collagen and exacerbating the combined effects of hepatocyte
injury, brosis and inammation, leading to NASH (Yang et al.,
2014). While the TGF-βpathway is central to liver brogenesis,
emerging proteome and transcriptome studies have suggested
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Acharya et al. Mechanisms of Liver Fibrosis
additional regulatory genes and pathways. These studies have
been carried out in animal models of NAFLD or NASH and
human liver biopsies obtained from patients. Comparative
transcriptomic studies between mouse models of NAFLD and
human liver biopsies obtained from NASH patients reveal major
differences between human NASH liver transcriptome and
mouse NAFLD transcriptomes even at severe stages (Teufel
et al., 2016). This suggests major pathophysiological
differences between human disease and animal models of the
disease and the need to design studies in humanized models of
disease or in liver organoid systems (Suppli et al., 2019). A meta-
analysis of transcriptomic studies carried out with human liver
biopsies suggests the upregulation of several genes within the
lipogenesis pathway (Table 2). Interestingly, genes such as
ACACA (Acetyl carboxylase 1) which catalyzes the synthesis of
malonyl CoA from acetyl CoA, the rate limiting step in fatty acid
biosynthesis and ACACB (Acetyl carboxylase 2) which regulates
fatty acid oxidation, are associated with NAFLD liver tissue
demonstrating the association of lipogenic functions within
the tissue with active disease (Table 2)(Widmer et al., 1996;
Locke et al., 2008). In several cases, NAFLD has been shown to be
linked to progression toward hepatocellular carcinoma. Recent
studies have led to the understanding that the evolution of
NAFLD to NASH and HCC is multifactorial and involves the
innate immune system to a great extent (Chen et al., 2019). Lipid
accumulation and mitochondrial dysfunction have been
identied as critical components of the pathways leading to
NAFLD (Margini and Dufour, 2016). Many new genes and
pathways have been implicated at every stage of NAFLD to
NASH to HCC progression (Figure 5). Regulation in PPAR-γ,
Insulin and p53-mediated signaling have been implicated in
NAFLD development, whereas signatures of inammatory
signaling such as Toll-like receptor (TLR) and Nucleotide-
binding, oligomerization domain (NOD) protein signaling
pathways, in addition to pathways reecting mitochondrial
dysfunction characterize NASH (Figure 5)(Ryaboshapkina
and Hammar, 2017).
There are only a limited number of proteomics studies in
human NAFLD. A comparative quantitative proteomics study
between NAFLD and Metabolic Healthy Obese (MHO)
individuals was carried out using liver tissue obtained during
surgery (Yuan et al., 2020). This study demonstrated the
relevance of PPAR signaling, ECM-receptor interaction and
oxidative phosphorylation in resisting NAFLD. Proteins
upregulated in NAFLD were involved in organization of the
ECM, and proteins downregulated in NAFLD were involved
in redox processes. A schematic of pathways relevant in
NAFLD progression, as gleaned from various omics
approaches is summarized in Figure 5.
Viral Hepatitis
In older children, autoimmune hepatitis and viral hepatitis are
the leading causes of liver brosis followed by cirrhosis. Viral
hepatitis can be caused by any one of the ve viruses: Hepatitis
A,B,C,D,andEofwhichAandEareusuallyacute,whileB,C,
and D are chronic (Zuckerman 1996). All hepatitis viruses are
infectious, while alcohol, other toxins and autoimmune
mediated hepatitis are usually non-infectious. HBV and HCV
lead to hepatic inammation (Gutierrez-Reyes et al., 2007).
Several viral components are known to induce cellular
damage in hepatocytes and liver constituents. For instance,
the HCV core protein in chronic infections is known to
interact with the TNF-αreceptors (TNFRSF1A) which
subsequently induces a pro-apoptotic signal in hepatocytes
(Zhu et al., 1998). Polymorphisms in TNFRSF1A have been
shown to be associated with HCV outcomes (Yue et al., 2021).
The HCV core protein is also known to interact with ApoA1and
ApoA2, thereby interfering with the assembly and secretion of
FIGURE 4 | The TGF-βsignaling pathway in hepatic stellate cells. TGF-βbinds to type II TGF-βrece ptor leading to receptor dimerization i.e. recruitment of the type I
TGF-βreceptor. The kinase domain of Type II TGF-βreceptor then phosphorylates the Ser residue of type I TGF-βreceptor. The phosphorylated receptor now recruits
R-SMAD, which binds to receptor through its N-terminal region and gets phos phorylated by the Type II receptor. The C-terminal of R-SMAD has a DNA binding domain
(DBD) that can act as a transcription factor. The co-SMAD now binds to R-SMAD and β-Importin binds to the dimer forming an oligomeric complex that guides the
R-SMAD and Co-SMAD into the nucleus. The dimer enters the nucleus and the DBD of SMAD now acts as transcription factor that can transcribe target genes.
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Acharya et al. Mechanisms of Liver Fibrosis
very low density lipoprotein (VLDL), thus cause the
accumulation of triglycerides in the liver through the
interaction of both viral and metabolic factors and
subsequent cell death (Gutierrez-Reyes et al., 2007).
Furthermore, the viral core protein as well as the HCV non-
structural protein 5A (NS5A) are known to cause mitochondrial
ROS production and cellular stress leading to cell death (Bataller
et al., 2004). Interestingly, HCV and NAFLD can co-exist and
have been shown to have a more rapid disease progression than
either disease alone (Patel and Harrison, 2012;Dyson et al.,
2014). About 50% of HCV patients have steatosis with
signicant brosis and the HCV genotype 3 is mainly
associated with the steatosis, however the exact mechanism
leading to steatosis in HCV patients is not fully elucidated.
The association of hepatitis B virus (HBV) infection with
NAFLD however, appears to be controversial. Some studies
suggest that HBV infection is protective against steatosis,
insulin resistance and metabolic syndrome (Morales et al.,
2017;Xiong et al., 2017) while others suggest that chronic
HBV infections can co-exist with NAFLD and can actively
worsen the disease (Zhang et al., 2020). The presence of
Hepatitis B protein X (HBx) in the cells has been shown to
increase the production of reactive oxygen species (ROS)
increasing the formation of lipids in the cells and therefore
HBx could be a risk factor for the development of NAFLD
(Wang et al., 2019). Therefore, an alternative mechanism by
which viral hepatitis can induce brosis is through their ability to
cause NAFLD.
TABLE 2 | Summary of pathways from transcriptomics analyses implicated in NAFLD
Gene Function/remarks References
LEP Leptin Anti-steatotic, but also a proinammatory and probrogenic action Polyzos et al. (2015)
PEMT Phosphatidylethanolamine
N-methyltransferase
Governs the secretion of hepatic triglycerides in the form of very low-density
lipoprotein
Tan et al. (2016)
PPAR-γ2Peroxisome proliferator activated receptor
gamma
Pparγ2 is expressed in the liver, specically in hepatocytes, and its expression level
positively correlates with fat accumulation induced by pathological conditions such
as obesity and diabetes
Lee et al. (2018)
TNF-αTumor necrosis factor Tumor necrosis factor (TNF)-αis associated with insulin resistance and systemic
inammatory responses
Seo et al. (2013)
PNPLA3 Patatin like phospholipase domain
containing 3
Polymorphisms in PNPLA3 have been linked to obesity and insulin sensitivity Chen et al. (2010)
CD14 CD14 molecule Upregulation of CD14 in liver cells show increased sensitivity to LPS, changes in
CD14 expression could represent a mechanism regulating liver sensitivity to LPS
toxicity
Satoh et al. (2013)
ACACA Acetyl-coa carboxylase αLow level is correlated with long time survival Liu et al, (2020)
ACACB Acetyl-coa carboxylase βInvolved in insulin signaling pathway and adipokine metabolic pathway Li et al. (2019)
ASPG Asparaginase The bacterial enzyme L-Asparaginase is a common cause of anti-neoplastic-
induced liver injury with occurrence of jaundice and marked steatosis
Kamal et al. (2019)
CCS Copper chaperone for superoxide
dismutase
CCS expression is regulated by copper by modulating its degradation by the 26S
proteosome
Bertinato and LAbbé
(2003)
CHEK1 Checkpoint kinase 1 This kinase is necessary to preserve genome integrity Zhang and Hunter (2014)
HDAC9 Histone deacetylase 9 Downregulation of HDAC9 decrease TGF-β1-induced brogenic gene expression
in hepatic stellate cells
Yang et al. (2017)
NADSYN1 NAD synthetase 1 Reduced NAD concentrations contribute to the dysmetabolic imbalance and
consequently to the pathogenesis of NAFLD
Guarino and Dufour (2019)
NHP2L1 Small nuclear ribonucleoprotein 13 This genes encodes a protein of the spliceosome complex Liu et al. (2020)
OAS3 2-5-oligoadenylate synthetase 3 OAS3 is an interferon-induced aniviral enzyme Zhang and Yu (2020)
PCNA Proliferating cell nuclear antigen PCNA encodes the protein which is found in the nucleus and is a cofactor of DNA
polymerase delta and involved in the RAD6-dependent DNA repair pathway in
response to DNA damage
Xing et al. (2018)
RPL10L Ribosomal protein L10 like The encoded protein shares sequence similarity with ribosomal protein L10 Liu et al. (2020)
RSL24D1 Ribosomal L24 domain containing 1 The encoded protein is involved in involved in the biogenesis of the early pre-60S
ribonucleoparticle
Xie et al. (2020)
SRC SRC proto-oncogene, non-receptor
tyrosine kinase
SRC is a proto-oncogene encoding a non-receptor tyrosine kinase Amanatidou and
Dedoussis (2021)
TOP2A DNA topoisomerase II alpha Regulates the topologic states of DNA and controls tumor cell response Wong et al. (2009)
TP53 Tumor protein p53 Induces apoptosis but the association between p53 and NAFLD remains
controversial, P53 plays an essential role in the pathogenesis of NAFLD, whereas
others have indicated that suppression of p53 activation aggravates liver steatosis
Yan et al. (2018)
TWISTNB RNA polymerase I subunit F This gene (i.e. TWIST Neighbor) is ubiquitous expressed in all tissues Liu et al. (2020)
UMPS Uridine monophosphate synthetase Lack of this gene results in reduced cell membrane stability Wortmann and Mayr
(2019)
HORMAD2 HORMA domain containing 2 Decreases with advancing brosis Liu et al. (2020)
LINC01554 Long intergenic non-protein coding RNA
1554
LINC01554, one kind of lncRNA, has been found specically enriched in liver tissue
and have strong association with pathogenesis and clinical evaluation of HCC
Ding et al. (2020)
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Acharya et al. Mechanisms of Liver Fibrosis
Parasitic Infections
The liver is capable of hosting a wide range of parasites which
vary in host cell requirement (extra or intracellular), sizes
(unicellular to multicellular) and potential harm to the host
cells or organs (Dunn, 2011). Parasites which have co-evolved
with humans through centuries, such as the malaria parasites
cause minimal injury to the host liver and move on to the blood
with ease (Acharya et al., 2017). However, some parasites can
cause injury to the cells of the liver and trigger the activation of
the brogenic pathway. Some of these parasites are discussed
below:
Leishmania is an intracellular protozoan parasite that infects
the reticuloendothelial system (RES) in the body, i.e., circulating
monocytes as well as tissue-resident macrophages (Magill et al.,
1993). Leishmaniasis is transmitted by the bite of infected
sandies (Dunn, 2011). Visceral leishmaniasis (kala-azar)
involves the RES infection of the visceral organs like the liver,
spleen, bone marrow and other lymph nodes. Kupffer cells, the
tissue resident macrophages of the liver, take up the amastigote
stage of Leishmania from circulating infected reticuloendothelial
cells. The parasite then replicates within the macrophages and
activates the host inammatory and Th1 and Th17 mediated
adaptive immune responses in immunocompetent individuals
(Pitta et al., 2009). Leishmaniasis is typically associated with
increased liver brosis (Melo et al., 2009). Leishmania
parasites have been shown to use host ECM components such
as bronectin and laminin to access Kupffer cells for infection
(Wyler et al., 1985;Wyler, 1987;Vannier-Santos et al., 1992;
Figueira et al., 2015). Visceral leishmaniasis has been frequently
studied in dogs as a model system. These studies suggest that dogs
infected with Leishmania have a signicantly higher level of
collagen and bronectin deposition (Melo et al., 2009). Intra-
lobular collagen deposition, appearance of MFBs and effacement
of the space of Disse are characteristic of overt Leishmania
infection in slightly or severely immunocompromized
individuals (Dunn, 2011). Leishmania associated brosis is
completely reversible once the parasitic infection has been
treated. However, since overt disease and severe brosis
usually occurs in immunocompromized individuals such as
those infected with HIV, relapses typically occur once
treatment ceases (Dunn, 2011).
Schistosomiasis is caused by Schistosoma species which are a
group of blood ukes belonging to the trematode or atworm
family (Andrade, 2009). It is prevalent mainly in the tropical and
sub-tropical regions of the world. Schistosoma use freshwater
snails as intermediate hosts, which release eggs into water bodies
which then come into contact with humans and infect them
(WHO. World Health Organization, 2021). Schistosomiasis can
be intestinal (wherein the liver is involved) or urogenital.
Intestinal schistosomiasis can be caused by many different
species such as Schistosoma mansoni (found in Africa, Middle
East, Caribbean, Brazil, Venezuela and Suriname), S. japonicum
(found in China, Indonesia and the Philippines), S. mekongi
(Cambodia, and the Lao Peoples Democratic Republic), S.
guineensis and S. intercalatum (found in the rain forests of
central Africa). Urogenital infection is caused by S.
hematobium (found in Africa, the Middle East and Corsica in
France) (WHO. World Health Organization, 2021).
Schistosoma mansoni are associated with liver brosis
(Andrade, 2009). Schistosome eggs are carried to the liver by
the portal vein and stop in the pre-sinusoidal vessels (Andrade,
2004). The development of severe schistosomiasis is thought to
have two components- (a) a major determinant is the high worm
load and, (b) a secondary determinant is thought to be genetic
predisposition. At low to moderate worm loads, many patients
are asymptomatic and the lesions heal automatically due to the
appropriate activation of T-cell mediated host immune responses
(Andrade, 2004). A high worm load is also associated with
damage to the portal vein and appearance of MFBs and
collagen deposition around the portal stem leading to portal
brosis called pipestem brosis (Andrade et al., 1999). Since all
infected individuals do not develop severe liver disease, or liver
FIGURE 5 | Summary of pathways that may be important in the progression of NAFLD to NASH. The transition from healthy to NAFLD involves the activation of
peroxisome proliferator activated receptor signaling, insulin signaling and p53 signaling whereas the switch to NASH involves activation of inammatory pathways such
as TLR and NOD like receptor mediated signaling, generation of intracellular oxidative stress and mitochondrial signaling.
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Acharya et al. Mechanisms of Liver Fibrosis
brosis, schistosomiasis linked liver brosis development is also
thought to have a genetic component. A metaanalysis of genetic
polymorphisms associated with severe liver disease and brosis in
schistosomiasis reveals several genetic polymorphisms (Dessein
et al., 2020). Several polymorphisms in genes related to the TGF-β
pathway were found to be associated with severe brosis in
schistosomiasis e.g., TGFBR1,TGFBR2,ACVRL1,SMAD3 and
SMAD9 (Dassein et al., 2020). Polymorphisms in the connective
tissue growth factor (CTGF) as well as the IL-22 pathway were
also observed. In addition, several associations have been
reported between severe hepatic brosis during Schistosomiasis
and genes encoding for IL-13, TNF-α, MAPKAP1, ST2, IL-10,
M1CA, HLADRB1, IL-4, ECP, and IFN-γ, have been reported
from various studies (Hirayama et al., 1998;Chevillard et al.,
2003;Eriksson et al., 2007;Gong et al., 2012;Silva et al., 2014;Zhu
et al., 2014;Long et al., 2015;Oliveira et al., 2015;Long et al., 2017;
Silva et al., 2017). These observations suggest that while infectious
agents such as schistosoma can drive hepatic brosis by
mediating tissue damage, genetic predispositions to TGF-β
pathway activation or a specicinammatory response may
make the hepatic environment conducive to brosis in the
presence of an infectious agent.
Fasciola hepatica, also known as the liver uke is also a
trematode parasite that infects humans (Machicado et al.,
2016). Fascioliasis is a neglected tropical disease. A recent
meta-analysis has found an association of Fasciola infections
with liver brosis, cirrhosis and hepatocellular carcinoma
(Machicado et al., 2016). The mechanism of brosis
development is thought to be due to the activation of HSCs by
parasite encoded cathepsins (Marcos et al., 2011). As with
schistosoma, worm-load seems to be an important
determinant of brosis. However, there are a very limited
number of studies available on the pathogenesis, molecular
epidemiology and prevalence of fascioliasis with liver brosis
and this area needs further investigation.
Cryptogenic Causes
Cryptogenic causes of liver brosis are cases with unknown
causes but it is believed that a high proportion of the
cryptogenic liver brosis cases could be linked to NAFLD or
NASH (Caldwell, 2010;Patel et al., 2020). Other causes could
include occult alcohol intake, viral hepatitis, autoimmune
hepatitis, biliary disease, vascular disease, celiac disease,
mitochondriopathies, systemic lupus erythematosus, Alstrom
syndrome, Apolipoprotein B with LDL cholesterol, and genetic
disorders such as short telomere syndrome, keratin 18 mutations
and glutathione-S-transferase mutations (Caldwell, 2010;Patel
et al., 2020).
SOLUBLE MEDIATORS IN LIVER FIBROSIS
The development of liver brosis occurs as a result of interaction
between several different cell types including hepatocytes, HSCs,
Kupffer cells, as well as inltrating immune cells. These inter-
cellular interactions involve several soluble and secreted
mediators which regulate inammatory pathways, chemotaxis
and HSC activation. Some of the known soluble mediators are
briey discussed below.
Cytokines and Chemokines
Cytokines are regulatory soluble small molecular weight proteins
or glycoproteins released by several cells and mediate interaction,
communication between different cell types. Cytokines play an
important role in the progress of liver brosis (Xu et al., 2012). In
liver they mediate the interactions of the various cell types and
contribute to either the production of proinammatory or
hepatoprotective responses (Kong et al., 2012). Cells of the
immune system such as Kupffer cells and neutrophils produce
many cytokines and chemokines that can affect the gene
expression, proliferation, contractility and activation of HSCs.
The interaction between HSCs and immune cells are
bidirectional, i.e., while immune cells produce cytokines to
activate HSCs. HSCs also regulate immune cell chemotaxis
and response by secreting soluble mediators themselves
(Weiskirchen, 2016). For instance, the pro-inammatory
cytokines TGF-αincreases HSC proliferation, TGF-βinhibits
HSC apoptosis and promotes ECM remodeling leading to a pro-
brogenic phenotype, TNF-αinhibits HSC apoptosis and induces
chemokines and ICAM-1 in HSCs (Maher, 2001). IL-4 in concert
with MMP-2 and ROS increase ECM synthesis and brosis. At
the same time, anti-brogenic cytokines are also released from
immune cells that can control the pro-brogenic HSC activation,
such as IL-10, IFN-α, IFN-γ. A balance of these factors results in a
net pro-or anti-brogenic effects on HSCs. The HSCs also
secretes several molecules which are instrumental in recruiting
immune cells at the site of activation such as M-CSF that causes
macrophage proliferation and maintenance, PAF, MIP-2 and
CINC/IL-8 which cause neutrophil chemotaxis and MCP-1
which recruits monocytes (Maher, 2001).
While activation of the TGF-βpathway is a central event in the
induction of hepatic brosis, HSC activation is regulated by other
pathways and molecular mechanisms as well, such as the Hippo
pathway and autophagy (Tsuchida and Friedman, 2017). The
Hippo signaling pathway is an evolutionarily conserved pathway
that derives its name from its key player, the protein kinase
Hippo, which is involved in the regulation of cell and organ size
(Saucedo and Edgar, 2007). However, Hippo pathway
components such as the transcriptional co-activator Yes-
associated protein 1 (YAP1) and the protein kinases
macrophage stimulating 1 (MST1) and MST 2 have been
shown to be important in initial HSC activation (Manmadhan
and Ehmer, 2019). Inhibition or silencing of YAP1, and
inactivation of MST1 and MST2 have been shown to have
therapeutic effects in mouse models of brosis but the human
clinical impact of such approaches is presently not known
(Manmadhan and Ehmer, 2019).
Chemokines are a subgroup of cytokines that have
chemotactic properties. They are synthesized by most liver
cells as well as by inltrating immune cells and their effects
depend on their local concentrations at the site of injury (Sahin
et al., 2010). Typically, chemokines bind G-protein coupled
receptors (GPCRs) and induce signaling in target cells
(Bonecchi et al., 2009). Stellate cells express several
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Acharya et al. Mechanisms of Liver Fibrosis
chemokines as well as chemokine receptors. HSCs have been
shown to secrete CCL2, CCL3, CCL5, CXCL1, CXCL8, CXCL9
and CXCL10 (Holt et al., 2009;Wasmuth et al., 2009;Zaldivar
et al., 2010;Marra and Tacke, 2014). Portal broblasts which are
involved in cholestastis-associated brosis are also capable of
secreting chemokines (Dranoff and Wells, 2010]. Targeting of
chemokines and chemokine receptors in experimental models of
brosis has been shown to control brosis and therefore warrants
further investigation as a potential therapeutic anti-brosis
strategy (Sahin et al., 2010).
In addition to these cytokines and chemokines, several
miRNA have been recently identied to be involved in the
HSC-immune cell cross-talk (Zhangdi et al., 2019).
Lipid Mediators
Lipid mediators in hepatic brosis are mainly studied in the
context of NAFLD and NASH (Liangpunsakul and Chalasani,
2019). Several different types of lipid species have been shown to
be associated with NAFLD such as saturated free fatty acids
(FFA), diacylglycerols, ceramides, lysophosphatidylcholine,
eicosanoids and free cholesterol (Feldstein et al., 2003;
Caballero et al., 2009;Gorden et al., 2011;Luukkonen et al.,
2016). Increased triglyceride accumulation is a hallmark of
NAFLD and is associated mainly with hepatic steatosis
(Yamaguchi et al., 2007). While triacylglycerol (TAG)
accumulation has not been found sufcient for causing insulin
resistance, excessive TAG accumulation can increase mechanical
pressure on hepatic sinusoids leading to the impairment of
hepatic blood ow, and generation of compensatory collateral
ow (Wanless and Shiota, 2004). Excessive amounts of free fatty
acids can act directly as TLR agonists in the liver or are taken up
by the liver, converted into lipotoxic intermediates that activate
the JNK, IKK pathway leading to cell injury, inammation and
apoptosis (Yu et al., 2002).
Extracellular Vesicles
Cellular injury to hepatocytes can lead to many outcomes. In
addition to hepatocyte cell death, injured and stressed
hepatocytes have been shown to release extracellular vesicles
(EV) (Ibrahim et al., 2016;Schattenberg and Lee, 2016). EV
are nanovesicles released by almost all cell types (Dooyle et al.,
2018). They constitute two major size categories, namely plasma
membrane derived microvesicles (501,000 nm) and endosome-
derived exosomes (30150 nm in diameter) as dened by the
International Society for Extracellular Vesicles(ISEV) and
according to the Minimal Information for Studies of
Extracellular Vesicles (MISEV) guidelines of 2014 (Lötvall
et al., 2014). In fact, lipid overload has been shown to activate
hepatocyte signaling through the death receptor 5 (DR5) followed
by release of hepatocyte-derived pro-inammatory EV
containing TNF-α(Cazanave et al., 2011). These EV activated
macrophage induced inammation leading to further cellular
injury and the development of NASH in experimental mouse
models (Cazanave et al., 2011). Administration of EV isolated
from high fat diet (HFD) mice into normal fed mice have been
shown to result in exacerbation of hepatic steatosis and
accumulation of activated myeloid cells in the liver through
the release of chemotactic EV (Ibrahim et al., 2016). In
addition to hepatocyte-derived EV, extra-hepatic EV have also
been implicated in the progression of NAFLD, NASH and
associated brosis (Srinivas et al., 2021). Due to their ability to
carry signal from one cell type to another, they can activate or
modulate target cell responses and are therefore an emerging
therapeutic targets in NAFLD and NASH.
Autophagy and Unfolded Protein Response
Autophagy in response to endoplasmic reticulum (ER) stress has
also been recognized as an activator of HSCs. Under normal
circumstances, autophagy is an important regulator of hepatic
homeostasis (Mallat et al., 2014). While normally, autophagy is
believed to have a protective effect on injured hepatocytes, recent
studies demonstrate that ER stress signals activate autophagy and
a probrogenic phenotype in HSCs (Mallat et al., 2014). HSC
activation is linked to increased ux in autophagy-related
metabolic pathways and inhibition of this process can prevent
HSC activation (Thoen et al., 2012). Similarly, there is evidence
that the accumulation of misfolded or unfolded proteins in the ER
triggering a process called unfolded protein response is a critical
feature during early activation of probrogenic cells such as HSCs
(Mannaerts et al., 2019), suggesting that the development of
interventions targeting the processes of autophagy or unfolded
proteins response might be effective in therapy of hepatic brosis.
CELLULAR MEDIATORS OF HEPATIC
FIBROSIS
The hallmark of hepatic brosis is the increased expression and
deposition of ECM compounds. There are different resident and
inltrating cells that can either be activated or produced by
progenitors that transform into a phenotype capable to
synthesize ECM. Each of these cell types have specic pro-
brogenic features and expression potential. Other cells invade
the inamed tissue and acquire a matrix-synthesizing phenotype
by reprogramming their cell fate (Figure 6). In most cases, TGF-β
regulated pathways contribute to the acquirement of brogenic
features. However, this might be due to the fact that several cell
types were only recently added to the list of probrogenic
progenitors and relevant signaling pathways still need to be
dened. In the following we will discuss how the different cells
contribute to hepatic brosis.
Hepatic Stellate Cells and Myobroblasts
HSCs reside in the perisinusoidal space between hepatocytes and
the sinusoids (i.e., the space of Disse). In the normal liver, these
cells exhibit a quiescent phenotype with the main known function
of storing vitamin A. During chronic hepatic disease, these cells
progressively lose their vitamin A, become activated and
transdifferentiate into brogenic MFBs that are supposed to be
the central cellular drivers of hepatic brosis in experimental and
human liver injury (Tsuchida and Friedman, 2017). In this
process, the induction of α-SMA is the most reliable marker
indicating cellular activation HSC. Fundamental fate tracing
experiments in mice have demonstrated that HSCs are the
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Acharya et al. Mechanisms of Liver Fibrosis
most important probrogenic cell type in the liver giving rise to
8296% of all MFBs in models of toxic, cholestatic and fatty liver
disease (Mederacke et al., 2013). HSCs typically express desmin
and vimentin, but other markers such as glial brillary acidic
protein (GFAP), lecithin retinol acyltransferase (LRAT),
synemin, platelet-derived growth factor receptor-β(PDGFRβ),
p75 neurotrophin receptor peptide (p75NTR), heart- and neural
crest derivatives-expressed 2 (HAND2), cytoglobin, and cysteine
and glycine-rich protein 2 (CRP2) have been discussed as HSC
specic markers within the liver (Weiskirchen et al., 2001;Suzuki
et al., 2008;Iwaisako et al., 2014;Kisseleva 2017;Tsuchida and
Friedman, 2017). However, the denition of general markers for
HSCs is rather complex because reporter microarray analysis,
gene mouse models and single cell RNA sequencing have
demonstrated the existence of distinct and functionally
relevant subsets of resting HSCs and activated MFBs, both in
vivo and in vitro (Magness et al., 2004;DAmbrosio et al., 2011; 9,;
Krenkel et al., 2019). Nevertheless, the expression of α-SMA and
collagen type I is signicantly increased during progression of
hepatic brosis conrming the view that MFBs are still most
likely the most relevant cell population contributing to hepatic
FIGURE 6 | Potential sources of extracellular matrix (ECM) producing cells in liver brosis. ECM producing cells during hepatic brosis can originate from many
sources. Hepatic stellate cells (HSCs) that transdifferenatiate into myobroblasts (MFBs), activated portal myobroblasts and activated resident broblasts are rich
sources of ECM. In addition, several other cell types that become activated, inltrate the liver, or originate by diverse transition processes are suitable to express large
quantities of ECM. Major pathways driving establishment of myobrogenic features are indicated for each progenitor. Abbreviations used are: ECM, extracellular
matrix; EGF, epithelial growth factor; EMT, epithelial-to-mesenchymal transition; FGF1/2, broblast growth factor 1/2; GLI1, glioma-associated oncogene homolog 1;
HGF, hepatocyte growth factor; IGF-1, insulin growth factor-1; IL, interleukin; MMT, mesothelial-to-mesenchymal transition; PDGF, platelet-derived growth factor; TGF-
α/β, transforming growth factor-α/β; VEGF, vascular endothelial growth factor. For details see text.
FIGURE 7 | Expression of brogenic markers in liver. The gure was
compiled using immunohistochemical data from the Human Protein Atlas
(www.proteinatlas.org/) (Uhlén et al., 2015). α-smooth muscle actin (α-SMA)
and collagen type 1α1 (COL1A1) proteins were stained in normal and
diseased livers.
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Acharya et al. Mechanisms of Liver Fibrosis
brosis (Figure 7). In addition, the expression of CRP2, Fibulin 2,
NGFR, PDGFRβ, Vimentin and many other genes is often used as
markers that become increased expressed during hepatic brosis
(Figure 8).
Recent studies have shown that there exists complex cellular
heterogeneity even within activated HSCs that convert into
collagen-secreting MFBs. Recent single-cell RNA sequencing
(scRNA-seq) studies in a CCl
4
-induced hepatic brosis model in
mice, clearly showed the presence of four sub-populations of
MFBs in the brotic liver that all express collagen but
differentially express chemokines (Krenkel et al., 2019).
Similarly, a very recent human liver scRNA-seq study has
revealed HSC heterogeneity along the porto-central axis of
thehealthyliverlobule(Valery et al., 2021). Two major HSC
sub-populations were obtained from the healthy human liver
lobules. One sub-population (HSC1) expressed high levels of the
cell surface proteoglycan glypican 3 (GPC3)andthe
neurotrophic tyrosine kinase receptor type 2 (NTRK2)along
with other commonly expressed HSC markers, whereas the
second sub-population (HSC2) expressed high levels of the
genes encoding for dopamine-norepinephrine converting
enzyme (DBH), hedgehog-interacting protein (HHIP), and
the G-protein coupled receptors (GPCRs) vasoactive
intestinal peptide receptor 1 (VIPR1), parathyroid hormone 1
receptor (PTH1R), receptor activity-modifying protein 1
(RAMP1), endothelin receptor type B (EDNRB), and
angiotensin receptor 1A (AGTR1A)(Valery et al., 2021). In
addition, beside the identication of novel quiescent markers
such as Quiescin Q6 sulfhydryl oxidase 1 (QSOX1)andsix-
transmembrane epithelial antigen of prostate 4 (STEAP4), these
scRNA-seq studies have also conrmed well-established HSC
marker including the regulator of G protein signaling (RGS5),
pleiotrophin (PTN), nerve growth factor receptor (NGFR),
lecithin retinol acyltransferase (LRAT), bulin 5 (FBLN5),
dihydrolipoamide branched-chain transacylase (DPT),
decorin (DCN), cytoglobin (CYGB), collectin 11 (COLEC11),
olfactomedin-like 3 (OLFML3), and tropomysosin 2 (TPM2),
respectively (Valery et al., 2021). All these ndings established
by scRNA-seq suggests this methodology as an emerging area
and promising experimental tool to reveal deeper insights into
HSC biology.
Portal Fibroblasts
In the normal liver, they encompass a quiescent phenotype with
a spindle-shaped broblastic phenotype that surrounds the
portal vein to maintain integrity of portal tract. In cholestatic
liver injury, portal MFBs are supposed to be a more important
source of activated MFBs than HSCs around proliferating bile
ducts (Tsuchida and Friedman, 2017). However, in contrast to
the well-characterized HSCs/MFBs the biology of these cells is
only partially known. Studies on rat portal MFB cell lines and
brotic mouse livers have shown that typical markers of this
probrogenic cells are elastin, type XV collagen α1,
ectonucleoside triphosphate diphosphohydrolase-2
(ENTPD2/CD39L1) and colin 1, while these cells are
negative for the HSC markers desmin, cytoglobin, and LRAT
(Iwaisako et al., 2014;Fausther et al., 2015). However, likewise
HSCs, these cells are positive for typical myobroblastic
markers including α-SMA, type I collagen α1, and tissue
inhibitor of metalloproteinase-1 (Fausther et al., 2015). Other
markers for portal MFBs were identied by
immunohistochemistry of brotic liver and FACS sorting of
liver cell preparations. These include Gremlin 1, Thy1/CD90,
Fibulin 2, mesothelin, asporin, and Mucin-16 (Iwaisako et al.,
2014;Kisseleva, 2017). Some of them are drastically induced
during progression of hepatic brosis, while their expression
signature might dependent on the hepatic insult analyzed
(Iwaisako et al., 2014).
FIGURE 8 | Additional markers of hepatic stellate cells and portal
myobroblasts. The gure was compiled from data deposited from Human
ProteinAtlas (www.proteinatlas.org/) (Uhlénet al., 2015). Immunohistochemistry
of the cysteine and glycine rich protein 2 (CRP2), Fibulin 2, nerve growth
factor receptor (NGFR),platelet-derivedgrowth factor-β(PDGFRβ) and Vimentin
in normal and diseased liver tissue. Liver damage is associated with increased
expression of these probrogenic markers. Image credit: Human Protein Atlas.
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Acharya et al. Mechanisms of Liver Fibrosis
Fibrocytes
Each organ has multiple populations of resident mesenchymal
cells capable of producing ECM. In the liver, HSCs and portal
broblasts are supposed to be the major cell types implicated in
the pathogenesis of liver brosis. Nevertheless, dependent of the
nature of hepatic insult, ECM producing cells may also originate
from many other sources. Fibrocytes are dened monocyte-
derived spindle-shaped cells having features of both
macrophages and broblasts (Reilkoff et al., 2011). Animal
experimentation using chimeric mice transplanted with donor
bone marrow from collagen α1(I)-GFP
+
reporter mice has shown
that collagen-producing brocytes are recruited from the bone
marrow to the damage liver tissue when recipient mice were
subjected to bile duct ligation (Kisseleva et al., 2006). Moreover,
when treated in culture with TGF-β1, these cells differentiated
into α-SMA and desmin positive collagen-producing MFBs.
Vascular Smooth Muscle Cells
Vascular smooth muscle cells (VSMCs) are integral components
of the blood vessel wall contributing to structural stability and
regulating vessel diameter. As such these contractile cells are
highly responsive toward vasoactive stimuli and contain a large
repertoire of specic contractile proteins facilitating their
dynamic phenotype (Metz et al., 2012). In response to injury,
VSMCs can shift from a contractile to a synthetic phenotype
characterized by increased expression of ECM compounds such
as collagen I and III and elevated expression of various non-
muscle myosin heavy chain isoforms (Metz et al., 2012). In
normal human liver, these cells are positive for α-SMA and
smoothelin representing a 59-kD cytoskeletal protein that is
found exclusively in contractile smooth muscle cells (Lepreux
et al., 2013). During the pathogenesis of advanced human liver
brosis, the cellular fraction of MFBs positive for both α-SMA
and smoothelin expanses to 510% suggesting a progressive
involvement of these resident cells in MFB recruitment
(Lepreux et al., 2013). Comparative transcriptome proling of
endothelial cells and VSMCs from canine vessels revealed an
enrichment of expression in genes associated with cytoskeleton
composition and actin lament organization including
transforming growth factor-β1(TGFB1), collagen type I α1
(COL1A1), nephroblastoma overexpressed gene (NOV),
Tenascin c (TNC), tissue factor pathway inhibitor 2 (TFPI2),
Tubulin α-4A (TUBA4A), Retinolbinding protein (RBP4),
insulin-like growth factor-binding protein 5 (IGFBP5), and
Cingulin-like 1 (CGNL1)(Oosterhoff et al., 2019). Single cell
transcriptomic further showed that VSMC in mouse and human
livers can be differentiated from other pro-brogenic cells of
mesenchymal origin (broblasts, HSCs) by their expression of
Calponin 1 (CNN1) or Myosin heavy chain 11 (MYH11)(Dobie
et al., 2019).
Bone Marrow-Derived Fibrocytes
The rst hints for a unique population of collagen-producing
brocytes derived from the bone marrow that could participate in
the pathogenesis of hepatic brosis were established in chimeric
mice transplanted with donor bone marrow from collagen
α1(I)-GFP
+
reporter mice (Kisseleva et al., 2006). In livers of
respective mice, a signicant increase in GFP
+
/CD45
+
positive
myobroblastic cells was observed when animals were subjected
to bile duct ligation, that however, were not positive for the
typical HSC markers α-SMA or vimentin underpinning their
lymphoid origin (Kisseleva et al., 2006). However, these cells
differentiated into α-SMA and desmin positive cells when
cultured in the presence of TGF-β1. A relevant functional
contribution of brocytes to the pathogenesis of hepatic
brosis was demonstrated in a mouse model in which
brocytes were specically depleted utilizing a herpes simplex
thymidine kinase/ganciclovir suicide approach in the
thioacetamide-induced liver brosis model (Hempel et al.,
2019). Although the depletion of brocytes resulted in
reduced deposition of brillar collagen, the antibrotic effect
was not accompanied by a reduction of MFBs. In the multidrug
resistance gene 2 knockout (Mdr2
/
)micespontaneously
developing cholestatic brosis, brocytes only minimally
contributed to the deposition of ECM in the injured livers
(Nishio et al., 2019). It will now be of fundamental interest,
to better dene the autocrine and paracrine functions of
brocytes during initiation and progression of hepatic
brosis in these and other models.
Hepatocytes
Hepatocytes are specialized epithelial cells making up 80% of the
total mass of the liver. They perform numerous vital functions,
including protein synthesis, metabolism of lipids and
carbohydrates, biotransformation and detoxication of
xenobiotics that enter the body. In addition, hepatocytes
synthesize and secrete bile and must therefore establish a
unique polarity in which apical (canalicular) and basolateral
(sinusoidal) plasma membranes are equipped with highly
specialized surface proteins, channels, and receptors (Schulze
et al., 2019). During liver injury these cells can contribute to
brogenesis by acquiring myobroblastic phenotypes/features
by undergoing a process termed epithelial-to-mesenchymal
transition (EMT) (Zeisberg et al., 2007). During this process
the cells downregulate epithelial features, lose their apical-basal
polarity, cell-cell adhesion properties and obtain migratory/
invasive properties, and acquire mesenchymal characteristics
allowing synthesizing ECM compounds (Yang et al., 2020).
Lineage-tracing experiments performed in transgenic mice in
which liver brosis were induced by repeated injections of
carbon tetrachloride demonstrated that up to 45% of
broblast-specic protein 1 (FSP1) positive broblasts
originated from hepatocytes via EMT (Zeisberg et al., 2007).
In line with the concept of EMT, primary mouse hepatocytes
transit in culture to FSP1 positive broblasts when cultured in
the presence of TGF-β1(Zeisberg et al., 2007). However, the
concept that brogenic cells capable to express type I collagen
can originate in vivo from hepatocytes was challenged by other
studies (Taura et al., 2010;Xie and Diehl, 2013). It was argued
that potential interpretational pitfalls may arise from the fact
that FSP1 is not only expressed in subsets of broblasts but is
also expressed by cells of the myeloid-monocytic lineage
(Scholten and Weiskirchen, 2011). However, the evidence for
and against EMT for the generation of myobroblastic cells
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Acharya et al. Mechanisms of Liver Fibrosis
from intrahepatic cells is still controversially discussed (Taura
et al., 2016;Munker et al., 2017;Chen et al., 2020).
Biliary Epithelial Cells
Similar to hepatocyte it was proposed that biliary epithelial cells
(i.e., cholangiocytes) can change their fate and transit to invasive
broblasts by EMT. In particular, in primary cirrhosis it was
demonstrated that bile duct epithelial cells express FSP1 and
vimentin as early markers of broblasts in the ductular reaction
(Robertson et al., 2007). In line, the stimulation of cultured
human cholangiocytes with TGF-βinduced expression of FSP1
and vimentin suggesting that these cells can contribute
signicantly to portal tract brosis (Rygiel et al., 2008). The
resulting cells formed in this localized EMT showed
coexpression of both cytokeratin-7 (CK-7) and FSP1
indicating that these cells have the capacity to migrate out of
the ductular structure (Rygiel et al., 2008). Several reports
suggested that sonic hedgehog signaling promotes EMT by
inducing myobroblast specic genes and repressing epithelial
genes during the pathogenesis of chronic biliary injury and
NAFLD (Omenetti et al., 2008;Syn et al., 2009). However, bile
duct ligation experiments performed in adult mice tagged with a
YFP reporter directed under regulatory control of the
cholangiocyte marker keratin 19 (K19) showed that
cholangiocytes that were positive for YFP revealed no
expression of EMT markers α-SMA, desmin, or FSP1
(Scholten et al., 2010).
Hepatic Progenitor Cells
The liver is the only visceral organ that can replace lost or
damaged tissue from the remaining tissue in a well-
orchestrated program, in which progenitor cells derived from
the biliary epithelium transdifferentiate to restore the hepatocyte
compartment (Michalopoulos 2013). Therefore, the occurrence
of resident hepatic progenitor cells (HPCs) was proposed that
should contain a dened cell fraction located in the canal of
Hering. The proposed cells should be characterized by a high
cellular plasticity and proliferation potential, the ability to
differentiate into hepatocytes and cholangiocytes, and to
mediate liver repopulation after injury (Li W et al., 2020).
However, also the conversion of hepatocytes to progenitor-like
cells has been documented in vitro (Li W et al., 2020). HPCs
isolated from chronically injured liver were shown to have
trilineage differentiation potential serving as progenitors for
hepatocytes, cholangiocytes and MFBs Sekiya et al., 2016).
Although the frequency of MFBs from HPCs was very low, it
can be speculated that HPCs can contribute to the MFB pool
during hepatic brogenesis (Sekiya et al., 2016).
Sinusoidal Endothelial Cells
Liver sinusoidal endothelial cells (LSEC) are a fenestrated cell
type without an organized basement membrane that forms the
predominant population in the hepatic sinusoid. In normal liver,
these cells form a selective barrier between the hepatocytes and
blood, possess a high endocytotic capacity allowing them to act as
an initial line of defense against invading pathogens, and are
critically involved in regulating vascular tone and permeability
(Hutchins et al., 2013). Under certain conditions these cells can
acquire an active phenotype characterized by swelling and
bulging of the cell body combined with enlargement of the
Golgi complex, increase of rough endoplasmic reticulum, and
formation of hemidesmosome-like structures that are hallmarks
of broblastic reticulum cells (Bardadin and Desmet, 1985).
During liver injury LSEC lose their fenestration, form a
continuous basal membrane, and develop inammatory and
brotic features, a process referred to as capillarization
(Baiocchini et al., 2019). Noteworthy, capillarized LSECs can
be an active contributor to the production of a brotic
environment during brogenesis by synthesis of collagen and
bronectin (Natarajan et al., 2017).
Mesothelial Cells
Mesothelial cells are specialized pavement-like cells forming a
protective layer of epithelial cells (i.e., the mesothelium) around
serous cavities and internal organs. These cells facilitate
transport of uid across these compartments and produce a
lubricating uid that is helpful in protecting the body against
infections (Mutsaers 2004). Observations from different animal
models and organ systems have shown that the adult
mesothelium of mice and humans contains a sub-population
of quiescent cells with stem-like properties (Koopmans and
Rinkevich, 2018). Upon peritoneal damage and appropriate
stimulus, these cells can be triggered to undergo a transition
process, termed mesothelial-to-mesenchymal transition
(MMT). The molecular reprogramming is associated with
morphological and functional changes and lead to cells
producing ECM compounds and pro-brogenic mediators
(Koopmans and Rinkevich, 2018). In line, TGF-β1in vitro
induced morphologic and functional reformation of
differentiated human mesothelial cells to MFBs that become
positive for α-SMA (Yang et al., 2003). In regard to liver
brogenesis, conditional cell lineage tracing in mice
conrmed that liver mesothelial cells can be driven by TGF-
βto generate both HSCs and MFBs depending on injury signals
in the liver (Li et al., 2013). While mesothelial cells preferentially
transit into HSCs in biliary brosis induced by bile duct ligation,
the cells majorly convert into MFBs in carbon tetrachloride-
induced brosis (Li Y et al., 2016). On the basis of lineage
tracing studies, it was supposed that mesothelial cells are
triggered by TGF-βto undergo MMT and contribute to the
MFBfractioninperitonealbrosis, in which up to 16.8% of all
MFBs were derived from peritoneal mesothelial cells (Lua et al.,
2015).
GLI1 Positive Perivascular Mesenchymal
Stem-like Cells
The glioma-associated oncogene homolog 1 (GLI1) belongs to
the family of three GLI C
2
H
2
-Kruppel type transcription factors
that contain ve zinc nger domains and either activate or
repress gene expression by binding to specic consensus DNA
sequences (Figure 9). Traditionally, GLI proteins are viewed as
downstream effectors of the Hedgehog (HH) signaling pathways,
but are now also known to be regulated transcriptionally and
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Acharya et al. Mechanisms of Liver Fibrosis
post-transcriptionally through non-canonical mechanisms
involving RAS-RAF-MEK-ERK and PI3K-AKT-mTOR (Dusek
and Hadden, 2021). This zinc nger protein was originally
identied as an oncogene that was amplied more than 50-
fold and highly expressed in some cases of malignant glioma
(Kinzler et al., 1987). GLI1 localize predominantly to the nucleus
(Figure 10) and bind the 9-base-pair consensus DNA 5-
GACCACCCA-3with high afnity (Kinzler and Vogelstein,
1990). It has turned out that the individual GLI proteins play
fundamental and distinct roles both in chronic inammation and
cancer. In some organs the lack of HH expression promotes
chronic inammation and tumor formation, while aberrantly
activated HH/GLI signaling is also capable to foster tumor
growth and simultaneously dampening inammation and
favoring immunosuppression (Grund-Gröschke et al., 2019).
Genetic lineage tracing analysis in mice demonstrated that
tissue-resident, but not circulating, GLI1 positive
mesenchymal-stem-cell-like cells can generate MFBs in
kidney, lung, liver, or heart after injury (Kramann et al.,
2015). Genetic ablation of GLI1 positive cells abolished bone
marrow brosis and rescued bone marrow failure (Schneider
et al., 2017). More recently it was demonstrated that the pro-
brogenic activity of osteopontin in promoting HSC activation
and ECM deposition during liver brogenesis is strongly
dependent on GLI1 function (Rao et al., 2019). In the human
HSC line LX-2, PAX6 binds to the promoter of the GLI1 gene,
thereby promoting brogenic activities and proliferation (Li C.
et al., 2020). In the same cell line, GLI1 was further shown to be
integrated in a complex network of Wnt/β-catenin, which
regulates cellular contraction (Zhang F. et al., 2020). However,
the signicance of GLI1 positive perivascular mesenchymal stem-
like cells for liver brogenesis is still unknown. Publicly available
FIGURE 9 | Crystal structure of the ve Zn ngers from human GLI1 in comple x with a high-afnity DNA binding site. Shown is a complex of a peptide derived from
the human GLI1 oncoprotein spanning region Glu 234 to Gly388 with a DNA fragment containing the specic binding site 5-GACCACCCA-3(underlined). Each of the
ve zinc ngers has a conserved sequence motif that is characterized by the consensus sequence X
3
-Cys-X
2-4
-Cys-X
12
-His-X
3-5
-His-X
4
(where X is any acid residue).
The structure has been determined at 2.6 Å resolution. Structure coordinates were taken from the PDB Protein Data Bank (access. no. 2GLI). For details see
(Pavletich and Pabo 1993).
FIGURE 10 | Expression of GLI1 in human bone osteocarcoma cell line U-2 OS. The cell line U-2 OS originating from human mesenchymal tumors express large
quantities of GLI1 (green), which is localized in the nucleus and the cytoplasm. Microtubuli (red) and nucleus (blue) are stained by a specic antibody or DAPI. The gure
was compiled using immunocytochemical data taken from the Human Protein Atlas v.20 (www.proteinatlas.org/) (Uhlén et al., 2015). They can be found at: https://www.
proteinatlas.org/ENSG00000111087-GLI1/cell#img.
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Acharya et al. Mechanisms of Liver Fibrosis
data obtained by single cell PCR shows that GLI1 mRNA
expression in normal human liver is rather low (<1 protein-
coding transcript per million) and restricted to some immune
cells and hepatocytes (Figure 11), while not found in smooth
muscle cells or endothelial cells. It will be now of particular
interest to document the existence of respective cells and to
clarify how these cells are triggered during hepatic brogenesis to
generate the proposed large fraction of MFBs.
MECHANISMS OF FIBROSIS REGRESSION
AND RESOLUTION
Liver brosis is potentially reversible (Ramachandran and
Iredale, 2012). Patients undergoing treatment for HCV
infection clearly demonstrate reversal of brosis upon
complete HCV negativity (Brenner, 2013). However, liver
brosis is reversible only in the early stages (Fibrosis grades
1 and 2). Once brosis crosses a threshold (Fibrosis grades 3
and 4), the brogenic type I collagen forms crosslinks and is
typically associated with cell damage and inammation,
making it harder to recover (Brenner, 2013). Two events
are critical in directing the liver pro-brogenic phenotype
to recovery - (i) Apoptosis of MFBs in the liver and, (ii)
switching of macrophages from a pro-inammatory to a
tissue resolution phenotype (Pellicoro et al., 2014). During
liver brogenesis, the ECM is extensively remodeled leading to
accumulation of proteases such as matrix metalloproteinases
(MMP)aswellascollagenases(Iredale, 2008). However, at the
same time, brotic liver also accumulates myobroblast-
derived tissue inhibitor of metalloproteinase 1 (TIMP1)
which prevents the action of MMPs and ECM turnover
(Iredale et al., 1996). As a result, there is an accumulation
of collagen and pro-brotic ECM. Over a period of time,
accumulation of a large number of crosslinked collagen and
elastin bers lead to sequestering of crosslinked bers within
the tissue beds, making them inaccessible for proteolytic
digestion (Issa et al., 2004). As the cross links increase, the
exposed bers also become less susceptible to digestion
themselves (Issa et al., 2004). The hallmark of a recovering
FIGURE 11 | GLI1 expression in liver (A) single cell PCR data shows that GLI1 mRNA expression in normal human liver is rather low (<1 protein-coding transcript
per million) and majorly restricted to a subpopulation of T-cells, B-cells and hepatocytes (B) Heatmap of marker gene expression in different hepatic cell types. The gure
was compiled using expression data from the Human Protein Atlas (www.proteinatlas.org/) (Uhlén et al., 2015). Abbreviations used are: pTPM, protein-coding transcript
per million; UMAP, uniform manifold approximation and projection.
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Acharya et al. Mechanisms of Liver Fibrosis
brotic liver is the termination of cellular injury followed by
the absence or disappearance of hepatic MFBs (Kisseleva et al.,
2012).Studiesshowthatatleast50%oftheactivatedMFBs
revert to less brogenic or quiescent HSCs (Kisseleva et al.,
2012;Troeger et al., 2012). The role of macrophages and the
trigger of switching from pro-inammatory to pro-resolution
macrophages during brosis is incompletely understood.
However, macrophages in the resolving brotic liver have
been shown to secrete increased levels of MMPs thereby
contributing to ECM reorganization (Li H et al., 2016).
Therefore, polarization of macrophages provides a
therapeutic opportunity for the resolution of liver brosis.
THERAPY OF HEPATIC FIBROSIS
Although numerous drugs have benecial anti-brotic effects
in vitro and in animal models, none of these drugs has been
ultimately shown to be efcacious in the clinic. Moreover, general
anti-brotic therapies are not available. Instead, clinicians and
professional associations have developed some clinical practice
guidelines and recommendations for etiology-specic
interventions. Most noticed are the guidelines published by the
American Association for the Study of Liver Diseases (AASLD)
and the European Association for the Study of Liver Diseases
(EASL) that both develop evidence-based clinical practice
guidelines on a regularly basis. These state-of-the-art
recommendations are intended to assist physicians and other
healthcare providers in the diagnosis and management of a
specic etiology of liver injury. As such they typically contain
information about disease denition, epidemiology, etiology, risk
factors, incidence, recommended tests and examinations for
disease detection, screening tools, preferred staging and
grading systems, therapy strategies, surveillance tests/intervals,
therapy outcome measures, prevention strategies, ongoing trials,
and much other supporting information. From the view of basic
scientists some generally applicable concepts should be effective
in the therapy of hepatic brosis. These include the withdrawal of
injurious stimuli, inhibition of ongoing hepatic damage,
deactivation and elimination of ECM-producing cells, removal
of superuous scar tissue, counteracting biological mediators
driving hepatic inammation and brogenesis, and restoring
the normal liver architecture (Figure 12). scRNA-seq and
genetic cell tracing experiments have shown that the
termination of hepatic brosis is associated with a reversal of
HSC activation and expression of different inactivation markers
(Troeger et al., 2012). However, reverted HSCs remain in a
primed state maintaining a higher responsiveness toward
brogenic stimuli.
Arrest of Chronic Liver Damage by Avoiding
or Eradication of Harmful Substances and
Replacement Therapies
As outlined, there are many genetic and environmental factors
that can cause hepatic brosis. Most frequent inherited disorders
FIGURE 12 | Potential therapeutic options for liver brosis. Based on the fact that hepatic brosis is driven by different mediators and pathways, there is a plenitude
of possibilities to interfere with this process. For more details see text or refer to (Schon et al., 2016;Weiskirchen 2016;Tacke and Weiskirchen , 2018;Weiskirchen et al.,
2018;Levada et al., 2019).
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Acharya et al. Mechanisms of Liver Fibrosis
associated with acute and chronic liver disease include
hemochromatosis, Wilson disease, α1-antitrypsin deciency,
and cystic brosis. In the case of hemochromatosis, excessive
iron can be removed from the body by regularly phlebotomy, or
alternatively iron chelating therapy (Murphree et al., 2020).
Wilson disease occurring as a consequence of impaired biliary
copper excretion can be effectively treated with the chelators
D-Penicillamine and trientine or by application of zinc
preparations interfering with the gastrointestinal uptake of
copper (Stremmel and Weiskirchen, 2021). Shortage of α1-
antitrypsin in the lung can be partially overcome by
intravenous replacement therapy, while this therapy is not
appropriate to people with liver disease. In respective patients,
there is emphasis on efforts to prevent progression of related liver
injury by reducing of modiable risk factors (overweight, tobacco,
alcohol, non-steroidal anti-inammatory drugs) or nally liver
transplantation that still remains the sole curative option
(Narayanan and Mistry, 2020). In cystic brosis resulting of
mutations in the gene encoding the cystic brosis
transmembrane conductance regulator (CFTR) the application
of Ursodeoxycholic acid (UDCA) is the mainstay of therapy. This
secondary bile acid is supposed to stimulate bile acid secretion,
but its efcacy is therapy of cystic brosis-related liver disease is
controversially discussed (Staufer, 2020). In addition, a large
number of compounds restoring CFTR protein function
(i.e., CFTR modulators) are actually under close investigation
(Staufer, 2020).
In regard to alcoholic liver disease, the abstinence from
drinking alcoholic beverages is quite the cornerstone of
therapy. However, several studies have shown that
glucocorticoids given alone or in combinations with
antioxidants are benecial to lower hepatic inammation
(Mitchell et al., 2020). In addition, drugs able to prevent
the development of steatosis, modulate innate immune
responses, targeting the microbiome, or stimulating liver
regeneration are investigated in many clinical studies
(Mitchell et al., 2020).
Antiviral treatment strategiesaresuitabletoreducethe
burden of chronic hepatitis B (HBV), hepatitis C virus (HCV),
or hepatitis D (HDV) infections. In particular, the
introduction of direct antiviral drugs offers nowadays a
very competent way to obtain viral clearance with sustained
virologic response rates greater than 95% (Do and Reau,
2020). Rigorous infant prophylaxis, early childhood and
adult immunization programs as well as vaccination of
high-risk individuals signicantly contribute to prevalence
of HBV transmission worldwide (Polaris Observatory
Collaborators, 2018). Established interferon-based therapies
are established and new encouraging drugs are currently
under clinical evaluationforthetreatmentofHDV(Koh
et al., 2019).
Autoimmune diseases of the liver typically affect either the
liver parenchyma, which is termed autoimmune hepatitis, or
alternatively the bile ducts provoking primary biliary
cholangitis (PBC) or primary sclerosing cholangitis (PSC)
(Weiskirchen, 2016). Immunosuppressive treatment
consisting of either corticosteroids alone or in combination
with the purine analog azathioprine is the recommended rst-
line medical treatment in autoimmune hepatitis (Tanaka,
2020).
There are some pharmacological approaches available for
the management of NAFLD and NASH, but an ultimate
therapy is still missing and actual guidelines presently only
recommend signicant changes in lifestyle and nutrition, in
particular weight loss and physical exercise (Drescher et al.,
2019). Nevertheless, there are several drugs currently at
various stages of development for the therapy of NASH,
possessing anti-inammatory activity, improve insulin
resistance, reduce de novo lipogenesis, modulate lipid
transport or oxidation, or evolve anti-apoptotic effects
(Tacke and Weiskirchen, 2018).
Liver brosis resulting from Schistosomiasis (S. mansoni and
S. japonicum) are presently treated with the pyrazinoisoquinoline
derivative praziquantel, while several vaccines that are urgently
needed are currently at differing phases of clinical development
and not yet been accepted for public use (McManus et al., 2020).
Antioxidants
As discussed, elevated quantities of reactive oxygen species
(ROS) are key drivers of hepatic inammation and brosis.
Under normal condition, ROS are required for many important
signaling processes, impact cell proliferation, contribute to
apoptotic pathways, and help phagocytic active cells to
destroy and eliminate pathogens (Luangmonkong et al.,
2018). They induce apoptosis and necrosis of parenchymal
cells (i.e., hepatocytes) resulting in the release of harmful
mediators (e.g., TGF-β,TNF-α), stimulate Kupffer cells to
produce probrogenic mediators, prompt recruitment of
circulating inammatory cells into the liver, and contribute
to the activation of HSCs (Weiskirchen, 2016). Therefore, an
imbalance between ROS production and degradation play an
important role in the pathogenesis of liver brosis.
Consequently, therapeutic interventions targeting elevated
cellular oxidative stress should be benecial for the treatment
of liver brosis (Luangmonkong et al., 2018). In regard to
therapy of liver brosis, many ROS inhibitors have been
tested successfully in pre-clinical animal models
(Weiskirchen, 2016). Most of these antioxidants are
scavengers that unspecic alleviate ROS accumulation, while
others are more selective by targeting dened molecular
pathways involved in ROS generation. In particular,
inhibitors of mitochondrial dysfunction (Coenenzyme !0,
Mitoquinone mesylate, NIM811), endoplasmic stress
(Glycerol phylbutyrate), NADPH oxidases (GKT137831,
Docosahexaenoic acid, losartan), and Toll-like receptors
(Curcumin, Quercetin, various probiotics, Bicyclol) have
attracted widespread attention in recent years
(Luangmonkong et al., 2018). Most promising are drugs that
interfere with the activity of different NADPH oxidase (NOX)
subtypes. In experimental models, both the deciency of NOX1
or NOX4 as well as the application of the dual NOX1/4 inhibitor
GKT137831 was effective in attenuation of carbon
tetrachloride-induced liver brosis (Lan et al., 2015).
Likewise, the NOX inhibitor apocynin was therapeutically
Frontiers in Pharmacology | www.frontiersin.org May 2021 | Volume 12 | Article 67164020
Acharya et al. Mechanisms of Liver Fibrosis