Cellular Mechanisms of Liver Fibrosis

1The liver is the largest solid organ in the human body, weighing about 1,200–1,500 g, and comprising about 1/50th of the total body weight in an adult. Understanding the complex architecture of the liver is key to understanding liver fibrosis and its consequences.

2The 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 Glisson’s capsule which is mainly composed of connective tissue. Within the Glisson’s 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 fibrosis and is completely damaged during cirrhosis (Figure 1A). 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. 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 detoxification of xenobiotics, urea cycle and the synthesis of plasma proteins. Hepatocytes are arranged in straight lines radiating out from the central vein toward the edge of the lobule. The space between the radially arranged files 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 flowing through the blood capillaries from the portal vein and the hepatic artery. Interactions between the parenchymal and non-parenchymal cells in this carefully preserved architecture are central to efficient functioning of the liver.

3Fibrogenesis is a normal wound healing response to tissue injury. All hepatocellular injuries activate the fibrogenic pathways. Once these pathways are activated, fibrogenic 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. During the encapsulation, there is an infiltration of immune cells that clear cellular debris and initiate tissue repair. The transition from a normal liver to fibrotic 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 fibronectin and laminin, type IV collagen (non-fibrogenic) 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 6–8 fold increase in the production of ECM components. Non-fibrogenic type IV collagen is replaced by fibrogenic type I and II collagen (Figure 1B). There is additional secretion of fibronectin, 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 fibrogenesis is intended at promoting wound healing, repeated injury and activation of the fibrogenic pathways result in a chronic activation of fibrogenesis. 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 surrounding the lobules which is the hallmark of fibrosis. 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 flow 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. Accumulation of type I collagen fibers leads to mechanical rigidity in the ECM which puts pressure on the blood vessels flowing through the liver. This leads to intrahepatic vasoconstriction and vascular resistance. Therefore, in a major vein like the hepatic portal vein, this leads to portal hypertension, which is a major clinical concern in liver fibrosis. 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. This further increases blood flow 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. These physiological processes are summarized in Figure 1B.

4Several genetic diseases predispose the liver to fibrosis. In all of these diseases, the initiation of fibrosis begins with tissue injury due to a consequence of the genetic defect followed by a fibrogenic wound healing response, as discussed above. Genetic causes for liver fibrosis 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 fibrosis, leading to cirrhosis. Most of these mutations affect many different cell types but predispose the individual to liver fibrosis and in some cases, liver cirrhosis. 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. In addition to the genetic mutations that predispose individuals to hepatic fibrosis that appear in childhood, mutations of the PNPLA3 gene have been described as a major predisposing factor in non-alcoholic fatty liver disease (NAFLD). PNPLA3 encodes for Patatin-like phospholipase domain-containing protein 3 or adiponutrin and is abundantly expressed in hepatocytes, adipocytes as well as HSCs. 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. 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%. Therefore, PNPLA3 gene is a strong predisposing genetic factor for hepatic fibrosis. Although the PNPLA3 protein has been shown to have triacylglycerol lipase and acylglycerol transacylase enzymatic activities, its exact role in hepatocytes have been controversial. Other studies have demonstrated a retinyl esterase activity for PNPLA3. HSCs are reservoirs for retinoic acid, which activate the retinoic acid receptor (RAR) mediated transcription which keeps fibrogenesis under control. 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 fibrogenesis in HSCs. However, it is now recognized that PNPLA3 has pleiotropic roles in the hepatocyte that are still under investigation such as in hepatocyte lipid droplet homeostasis, HSC quiescence and proliferation regulation. As several roles for PNPLA3 are suggested, PNPLA3 might be a good therapeutic target to control NAFLD related fibrosis and disease progression.

5Excessive and continued alcohol intake over large periods of time, i.e., alcohol abuse, can lead to liver fibrosis followed by cirrhosis and liver cancer. Alcoholic liver disease (ALD) comprises a spectrum of liver disorders ranging from fatty liver, steatosis, fibrosis with varying degrees of inflammation, cirrhosis. Alcohol abuse contributes to almost 50% of chronic liver disease related deaths globally. 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. Alcohol cannot be stored and needs to undergo obligatory oxidation which occurs predominantly in the liver (Figure 2). The first 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. 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. 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. 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. The second major pathway for ethanol metabolism is via the inducible cytochrome P450 CYP1E2, also known as the microsomal ethanol oxidizing system (MEOS). This is located in the smooth endoplasmic reticulum of hepatocytes. In normal people with average to below average alcohol consumption, MEOS forms a minor pathway for intracellular alcohol metabolism. 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. 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 inflammation leading to alcoholic hepatitis which, in itself can be fatal. Often, these processes occur hand in hand. Cellular depletion of glutathione has an additional consequence. Glutathione is required for the detoxification of several drugs including acetaminophen. In the hepatocytes, acetaminophen is modified to form a cytotoxic metabolite known as N-acetyl-p-benzoquinone imine (NAPQI) via CYP2E1. Conjugation of NAPQI to glutathione results in an S-glutathione product that detoxifies 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. Long term alcohol use induces CYP2E1 and therefore facilitates rapid NAPQI formation when the liver encounters acetaminophen. At the 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 detoxification 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 fibrogenic pathway leading to hepatic fibrosis, cirrhosis and hepatocellular carcinoma.

6Drugs induce hepatic fibrosis by causing drug-induced liver injury (DILI) that causes the initiation of fibrogenic 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). 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 detoxification of xenobiotic compounds that enter our circulation either through diet or through intravenous drug usage. Detoxification mechanisms in the liver mainly involve the cytochrome P450 family (CYP gene families CYP1, CYP2, CYP3) (Figure 3). Cytochrome P450s are a group of heme proteins that are involved in the initial detoxification reactions of small molecules such as dietary and physiological metabolites, as well as drugs. The expression of the CYP genes is influenced by several factors such as age, sex, promoter polymorphisms, cytokines, xenobiotic compounds and hormones, to name a few. Cytochrome P450 mainly carry out a monooxygenation reaction and carry oxidation of drugs/xenobiotic compounds. This can either convert the molecule into an inert or bioactive molecule.

7Bioactive 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 influence the final outcome of drug intake such as age, gender, comorbidities, intake of alcohol, other drugs or herbal preparations and polymorphisms of the CYP genes. The exact mechanism of DILI in specific cases 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 profiles. 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 fibrogenic responses as described above.

8Cholestasis is emerging as a leading cause for liver injury and fibrosis. 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. The pathogenesis of cholestasis is unclear but is believed to have an autoimmune component to it. I primary sclerosing cholangitis (PSC) several strictures appear around the bile ducts and cause bile duct injury. This activates the portal fibroblasts around the bile duct, which then differentiate into collagen secreting myofibroblasts (MFBs) similar to those derived from HSC activation. Recent studies have shown that fibrogenic MFBs have inherent heterogeneity and can be derived from both HSCs and portal fibroblasts. PSC has been shown to be associated with a varied manifestation of other diseases such as inflammatory bowel disease, cholangiocarcinoma, high IgG4 levels, autoimmune hepatitis and colonic neoplasia. Due to its association with autoimmune responses, PSC is thought to involve a genetic predisposition which is activated by an as yet unidentified environmental trigger such as gut dysbiosis. Although PSC is traditionally recognized as a rare disease, its incidence is on the rise due to an increase in unknown environmental triggers. 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 fibroblasts and consequent fibrogenesis.

9The metabolic syndrome is a group of associated diseases that increase cardiovascular risk factors and are linked with obesity and type 2 diabetes mellitus. Liver manifestations of the metabolic syndrome result in NAFLD. 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%. NAFLD is linked to increased risk of hepatic fibrosis, hepatocellular carcinoma and mortality due to cardiovascular disease. The more severe subtype of NAFLD is NASH, which has a global prevalence of about 2–6% and which is associated with severe hepatic inflammation, fibrosis leading to cirrhosis and HCC as well as end stage liver disease. 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 9–32% in the general population and about 53% in obese individuals. 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 profibrogenic MFBs, through the activation of the TGF-β pathway (Figure 4). TGF-β is a pleiotropic cytokine and is involved in various cellular processes like cell proliferation, survival, angiogenesis, differentiation, and the wound healing response. TGF-β binds to the TGF-β receptor 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 specifically respond to the TGF-β receptor modulation. Phosphorylated SMADs subsequently translocate into the nucleus and control the expression of the TGF-β regulated target genes (Figure 4). The activation of HSCs via TGF-β plays a major role in the advanced NAFLD in both experimental animal models, as well as in human liver injury. In addition to HSC activation, TGF-β signaling followed by SMAD phosphorylation is known to cause hepatocyte death driving progression to NASH. Hepatocyte death via TGF-β signaling is accompanied by generation of reactive oxygen species as well as lipid accumulation in hepatocytes. Activation of the TGF-β pathway also leads to HSC differentiation into MFBs leading to formation of fibrillar collagen and exacerbating the combined effects of hepatocyte injury, fibrosis and inflammation, leading to NASH. While the TGF-β pathway is central to liver fibrogenesis, emerging proteome and transcriptome studies have suggested 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. 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. 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). 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. Lipid accumulation and mitochondrial dysfunction have been identified as critical components of the pathways leading to NAFLD. 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 inflammatory signaling such as Toll-like receptor (TLR) and Nucleotide-binding, oligomerization domain (NOD) protein signaling pathways, in addition to pathways reflecting mitochondrial dysfunction characterize NASH (Figure 5).

10There 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. 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.

11In older children, autoimmune hepatitis and viral hepatitis are the leading causes of liver fibrosis followed by cirrhosis. Viral hepatitis can be caused by any one of the five viruses: Hepatitis A, B, C, D, and E of which A and E are usually acute, while B, C, and D are chronic. All hepatitis viruses are infectious, while alcohol, other toxins and autoimmune mediated hepatitis are usually non-infectious. HBV and HCV lead to hepatic inflammation. 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-α receptors (TNFRSF1A) which subsequently induces a pro-apoptotic signal in hepatocytes. Polymorphisms in TNFRSF1A have been shown to be associated with HCV outcomes. The HCV core protein is also known to interact with ApoA1and ApoA2, thereby interfering with the assembly and secretion of 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. 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. Interestingly, HCV and NAFLD can co-exist and have been shown to have a more rapid disease progression than either disease alone. About 50% of HCV patients have steatosis with significant fibrosis 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.

12The 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 while others suggest that chronic HBV infections can co-exist with NAFLD and can actively worsen the disease. 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. Therefore, an alternative mechanism by which viral hepatitis can induce fibrosis is through their ability to cause NAFLD.

13The 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. 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. However, some parasites can cause injury to the cells of the liver and trigger the activation of the fibrogenic pathway. Some of these parasites are discussed below:

14Leishmania is an intracellular protozoan parasite that infects the reticuloendothelial system (RES) in the body, i.e., circulating monocytes as well as tissue-resident macrophages. Leishmaniasis is transmitted by the bite of infected sandflies. 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 inflammatory and Th1 and Th17 mediated adaptive immune responses in immunocompetent individuals. Leishmaniasis is typically associated with increased liver fibrosis. Leishmania parasites have been shown to use host ECM components such as fibronectin and laminin to access Kupffer cells for infection. Visceral leishmaniasis has been frequently studied in dogs as a model system. These studies suggest that dogs infected with Leishmania have a significantly higher level of collagen and fibronectin deposition. 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. Leishmania associated fibrosis is completely reversible once the parasitic infection has been treated. However, since overt disease and severe fibrosis usually occurs in immunocompromized individuals such as those infected with HIV, relapses typically occur once treatment ceases.

15Schistosomiasis is caused by Schistosoma species which are a group of blood flukes belonging to the trematode or flatworm family. 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. 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 People’s 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).

16Schistosoma mansoni are associated with liver fibrosis. Schistosome eggs are carried to the liver by the portal vein and stop in the pre-sinusoidal vessels. 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. 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 fibrosis called pipestem fibrosis. Since all infected individuals do not develop severe liver disease, or liver fibrosis, schistosomiasis linked liver fibrosis development is also thought to have a genetic component. A metaanalysis of genetic polymorphisms associated with severe liver disease and fibrosis in schistosomiasis reveals several genetic polymorphisms. Several polymorphisms in genes related to the TGF-β pathway were found to be associated with severe fibrosis in schistosomiasis e.g., TGFBR1, TGFBR2, ACVRL1, SMAD3 and SMAD9. 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 fibrosis 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. These observations suggest that while infectious agents such as schistosoma can drive hepatic fibrosis by mediating tissue damage, genetic predispositions to TGF-β pathway activation or a specific inflammatory response may make the hepatic environment conducive to fibrosis in the presence of an infectious agent.

17Fasciola hepatica, also known as the liver fluke is also a trematode parasite that infects humans. Fascioliasis is a neglected tropical disease. A recent meta-analysis has found an association of Fasciola infections with liver fibrosis, cirrhosis and hepatocellular carcinoma. The mechanism of fibrosis development is thought to be due to the activation of HSCs by parasite encoded cathepsins. As with schistosoma, worm-load seems to be an important determinant of fibrosis. However, there are a very limited number of studies available on the pathogenesis, molecular epidemiology and prevalence of fascioliasis with liver fibrosis and this area needs further investigation.

18Cryptogenic causes of liver fibrosis are cases with unknown causes but it is believed that a high proportion of the cryptogenic liver fibrosis cases could be linked to NAFLD or NASH. 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.

19The development of liver fibrosis occurs as a result of interaction between several different cell types including hepatocytes, HSCs, Kupffer cells, as well as infiltrating immune cells. These inter-cellular interactions involve several soluble and secreted mediators which regulate inflammatory pathways, chemotaxis and HSC activation. Some of the known soluble mediators are briefly discussed below.

20Cytokines 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 fibrosis. In liver they mediate the interactions of the various cell types and contribute to either the production of proinflammatory or hepatoprotective responses. 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. For instance, the pro-inflammatory cytokines TGF-α increases HSC proliferation, TGF-β inhibits HSC apoptosis and promotes ECM remodeling leading to a pro-fibrogenic phenotype, TNF-α inhibits HSC apoptosis and induces chemokines and ICAM-1 in HSCs. IL-4 in concert with MMP-2 and ROS increase ECM synthesis and fibrosis. At the same time, anti-fibrogenic cytokines are also released from immune cells that can control the pro-fibrogenic HSC activation, such as IL-10, IFN-α, IFN-γ. A balance of these factors results in a net pro-or anti-fibrogenic 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.

21While activation of the TGF-β pathway is a central event in the induction of hepatic fibrosis, HSC activation is regulated by other pathways and molecular mechanisms as well, such as the Hippo pathway and autophagy. 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. 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. Inhibition or silencing of YAP1, and inactivation of MST1 and MST2 have been shown to have therapeutic effects in mouse models of fibrosis but the human clinical impact of such approaches is presently not known.

22Chemokines are a subgroup of cytokines that have chemotactic properties. They are synthesized by most liver cells as well as by infiltrating immune cells and their effects depend on their local concentrations at the site of injury. Typically, chemokines bind G-protein coupled receptors (GPCRs) and induce signaling in target cells. Stellate cells express several chemokines as well as chemokine receptors. HSCs have been shown to secrete CCL2, CCL3, CCL5, CXCL1, CXCL8, CXCL9 and CXCL10. Portal fibroblasts which are involved in cholestastis-associated fibrosis are also capable of secreting chemokines. Targeting of chemokines and chemokine receptors in experimental models of fibrosis has been shown to control fibrosis and therefore warrants further investigation as a potential therapeutic anti-fibrosis strategy.

23In addition to these cytokines and chemokines, several miRNA have been recently identified to be involved in the HSC-immune cell cross-talk.

24Lipid mediators in hepatic fibrosis are mainly studied in the context of NAFLD and NASH. 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. Increased triglyceride accumulation is a hallmark of NAFLD and is associated mainly with hepatic steatosis. While triacylglycerol (TAG) accumulation has not been found sufficient for causing insulin resistance, excessive TAG accumulation can increase mechanical pressure on hepatic sinusoids leading to the impairment of hepatic blood flow, and generation of compensatory collateral flow. 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, inflammation and apoptosis.

25Cellular 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). EV are nanovesicles released by almost all cell types. They constitute two major size categories, namely plasma membrane derived microvesicles (50–1,000 nm) and endosome-derived exosomes (30–150 nm in diameter) as defined by the International Society for Extracellular Vesicles” (ISEV) and according to the Minimal Information for Studies of Extracellular Vesicles (MISEV) guidelines of 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-inflammatory EV containing TNF-α. These EV activated macrophage induced inflammation leading to further cellular injury and the development of NASH in experimental mouse models. 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. In addition to hepatocyte-derived EV, extra-hepatic EV have also been implicated in the progression of NAFLD, NASH and associated fibrosis. 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.

26Autophagy 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. While normally, autophagy is believed to have a protective effect on injured hepatocytes, recent studies demonstrate that ER stress signals activate autophagy and a profibrogenic phenotype in HSCs. HSC activation is linked to increased flux in autophagy-related metabolic pathways and inhibition of this process can prevent HSC activation. 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 profibrogenic cells such as HSCs, suggesting that the development of interventions targeting the processes of autophagy or unfolded proteins response might be effective in therapy of hepatic fibrosis.

27The hallmark of hepatic fibrosis is the increased expression and deposition of ECM compounds. There are different resident and infiltrating 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 specific pro-fibrogenic features and expression potential. Other cells invade the inflamed 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 fibrogenic features. However, this might be due to the fact that several cell types were only recently added to the list of profibrogenic progenitors and relevant signaling pathways still need to be defined. In the following we will discuss how the different cells contribute to hepatic fibrosis.

28HSCs 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 fibrogenic MFBs that are supposed to be the central cellular drivers of hepatic fibrosis in experimental and human liver injury. 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 most important profibrogenic cell type in the liver giving rise to 82–96% of all MFBs in models of toxic, cholestatic and fatty liver disease. HSCs typically express desmin and vimentin, but other markers such as glial fibrillary 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 specific markers within the liver. However, the definition 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 (;; 9,; ). Nevertheless, the expression of α-SMA and collagen type I is significantly increased during progression of hepatic fibrosis confirming the view that MFBs are still most likely the most relevant cell population contributing to hepatic fibrosis (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 fibrosis (Figure 8).

29Recent 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 CCl4-induced hepatic fibrosis model in mice, clearly showed the presence of four sub-populations of MFBs in the fibrotic liver that all express collagen but differentially express chemokines. Similarly, a very recent human liver scRNA-seq study has revealed HSC heterogeneity along the porto-central axis of the healthy liver lobule. 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) and the 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). In addition, beside the identification of novel quiescent markers such as Quiescin Q6 sulfhydryl oxidase 1 (QSOX1) and six-transmembrane epithelial antigen of prostate 4 (STEAP4), these scRNA-seq studies have also confirmed well-established HSC marker including the regulator of G protein signaling (RGS5), pleiotrophin (PTN), nerve growth factor receptor (NGFR), lecithin retinol acyltransferase (LRAT), fibulin 5 (FBLN5), dihydrolipoamide branched-chain transacylase (DPT), decorin (DCN), cytoglobin (CYGB), collectin 11 (COLEC11), olfactomedin-like 3 (OLFML3), and tropomysosin 2 (TPM2), respectively. All these findings established by scRNA-seq suggests this methodology as an emerging area and promising experimental tool to reveal deeper insights into HSC biology.

30In the normal liver, they encompass a quiescent phenotype with a spindle-shaped fibroblastic 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. 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 fibrotic mouse livers have shown that typical markers of this profibrogenic cells are elastin, type XV collagen α1, ectonucleoside triphosphate diphosphohydrolase-2 (ENTPD2/CD39L1) and cofilin 1, while these cells are negative for the HSC markers desmin, cytoglobin, and LRAT. However, likewise HSCs, these cells are positive for typical myofibroblastic markers including α-SMA, type I collagen α1, and tissue inhibitor of metalloproteinase-1. Other markers for portal MFBs were identified by immunohistochemistry of fibrotic liver and FACS sorting of liver cell preparations. These include Gremlin 1, Thy1/CD90, Fibulin 2, mesothelin, asporin, and Mucin-16. Some of them are drastically induced during progression of hepatic fibrosis, while their expression signature might dependent on the hepatic insult analyzed.

31Each organ has multiple populations of resident mesenchymal cells capable of producing ECM. In the liver, HSCs and portal fibroblasts are supposed to be the major cell types implicated in the pathogenesis of liver fibrosis. Nevertheless, dependent of the nature of hepatic insult, ECM producing cells may also originate from many other sources. Fibrocytes are defined monocyte-derived spindle-shaped cells having features of both macrophages and fibroblasts. Animal experimentation using chimeric mice transplanted with donor bone marrow from collagen α1(I)-GFP+ reporter mice has shown that collagen-producing fibrocytes are recruited from the bone marrow to the damage liver tissue when recipient mice were subjected to bile duct ligation. Moreover, when treated in culture with TGF-β1, these cells differentiated into α-SMA and desmin positive collagen-producing MFBs.

32Vascular 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 specific contractile proteins facilitating their dynamic phenotype. 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. 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. During the pathogenesis of advanced human liver fibrosis, the cellular fraction of MFBs positive for both α-SMA and smoothelin expanses to 5–10% suggesting a progressive involvement of these resident cells in MFB recruitment. Comparative transcriptome profiling of endothelial cells and VSMCs from canine vessels revealed an enrichment of expression in genes associated with cytoskeleton composition and actin filament 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), Retinol–binding protein (RBP4), insulin-like growth factor-binding protein 5 (IGFBP5), and Cingulin-like 1 (CGNL1). Single cell transcriptomic further showed that VSMC in mouse and human livers can be differentiated from other pro-fibrogenic cells of mesenchymal origin (fibroblasts, HSCs) by their expression of Calponin 1 (CNN1) or Myosin heavy chain 11 (MYH11).

33The first hints for a unique population of collagen-producing fibrocytes derived from the bone marrow that could participate in the pathogenesis of hepatic fibrosis were established in chimeric mice transplanted with donor bone marrow from collagen α1(I)-GFP+ reporter mice. In livers of respective mice, a significant increase in GFP+/CD45+ positive myofibroblastic 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. However, these cells differentiated into α-SMA and desmin positive cells when cultured in the presence of TGF-β1. A relevant functional contribution of fibrocytes to the pathogenesis of hepatic fibrosis was demonstrated in a mouse model in which fibrocytes were specifically depleted utilizing a herpes simplex thymidine kinase/ganciclovir suicide approach in the thioacetamide-induced liver fibrosis model. Although the depletion of fibrocytes resulted in reduced deposition of fibrillar collagen, the antifibrotic effect was not accompanied by a reduction of MFBs. In the multidrug resistance gene 2 knockout (Mdr2 −/−) mice spontaneously developing cholestatic fibrosis, fibrocytes only minimally contributed to the deposition of ECM in the injured livers. It will now be of fundamental interest, to better define the autocrine and paracrine functions of fibrocytes during initiation and progression of hepatic fibrosis in these and other models.

34Hepatocytes 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 detoxification 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. During liver injury these cells can contribute to fibrogenesis by acquiring myofibroblastic phenotypes/features by undergoing a process termed epithelial-to-mesenchymal transition (EMT). 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. Lineage-tracing experiments performed in transgenic mice in which liver fibrosis were induced by repeated injections of carbon tetrachloride demonstrated that up to 45% of fibroblast-specific protein 1 (FSP1) positive fibroblasts originated from hepatocytes via EMT. In line with the concept of EMT, primary mouse hepatocytes transit in culture to FSP1 positive fibroblasts when cultured in the presence of TGF-β1. However, the concept that fibrogenic cells capable to express type I collagen can originate in vivo from hepatocytes was challenged by other studies. It was argued that potential interpretational pitfalls may arise from the fact that FSP1 is not only expressed in subsets of fibroblasts but is also expressed by cells of the myeloid-monocytic lineage. However, the evidence for and against EMT for the generation of myofibroblastic cells from intrahepatic cells is still controversially discussed.

35Similar to hepatocyte it was proposed that biliary epithelial cells (i.e., cholangiocytes) can change their fate and transit to invasive fibroblasts by EMT. In particular, in primary cirrhosis it was demonstrated that bile duct epithelial cells express FSP1 and vimentin as early markers of fibroblasts in the ductular reaction. In line, the stimulation of cultured human cholangiocytes with TGF-β induced expression of FSP1 and vimentin suggesting that these cells can contribute significantly to portal tract fibrosis. 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. Several reports suggested that sonic hedgehog signaling promotes EMT by inducing myofibroblast specific genes and repressing epithelial genes during the pathogenesis of chronic biliary injury and NAFLD. 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.

36The 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. Therefore, the occurrence of resident hepatic progenitor cells (HPCs) was proposed that should contain a defined 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. However, also the conversion of hepatocytes to progenitor-like cells has been documented in vitro. HPCs isolated from chronically injured liver were shown to have trilineage differentiation potential serving as progenitors for hepatocytes, cholangiocytes and MFBs ). Although the frequency of MFBs from HPCs was very low, it can be speculated that HPCs can contribute to the MFB pool during hepatic fibrogenesis.

37Liver 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. 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 fibroblastic reticulum cells. During liver injury LSEC lose their fenestration, form a continuous basal membrane, and develop inflammatory and fibrotic features, a process referred to as capillarization. Noteworthy, capillarized LSECs can be an active contributor to the production of a fibrotic environment during fibrogenesis by synthesis of collagen and fibronectin.

38Mesothelial 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 fluid across these compartments and produce a lubricating fluid that is helpful in protecting the body against infections. 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. 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-fibrogenic mediators. In line, TGF-β1 in vitro induced morphologic and functional reformation of differentiated human mesothelial cells to MFBs that become positive for α-SMA. In regard to liver fibrogenesis, conditional cell lineage tracing in mice confirmed that liver mesothelial cells can be driven by TGF-β to generate both HSCs and MFBs depending on injury signals in the liver. While mesothelial cells preferentially transit into HSCs in biliary fibrosis induced by bile duct ligation, the cells majorly convert into MFBs in carbon tetrachloride-induced fibrosis. On the basis of lineage tracing studies, it was supposed that mesothelial cells are triggered by TGF-β to undergo MMT and contribute to the MFB fraction in peritoneal fibrosis, in which up to 16.8% of all MFBs were derived from peritoneal mesothelial cells.

39The glioma-associated oncogene homolog 1 (GLI1) belongs to the family of three GLI C2H2-Kruppel type transcription factors that contain five zinc finger domains and either activate or repress gene expression by binding to specific 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 post-transcriptionally through non-canonical mechanisms involving RAS-RAF-MEK-ERK and PI3K-AKT-mTOR. This zinc finger protein was originally identified as an oncogene that was amplified more than 50-fold and highly expressed in some cases of malignant glioma. GLI1 localize predominantly to the nucleus (Figure 10) and bind the 9-base-pair consensus DNA 5′-GACCACCCA-3′ with high affinity. It has turned out that the individual GLI proteins play fundamental and distinct roles both in chronic inflammation and cancer. In some organs the lack of HH expression promotes chronic inflammation and tumor formation, while aberrantly activated HH/GLI signaling is also capable to foster tumor growth and simultaneously dampening inflammation and favoring immunosuppression. 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. Genetic ablation of GLI1 positive cells abolished bone marrow fibrosis and rescued bone marrow failure. More recently it was demonstrated that the pro-fibrogenic activity of osteopontin in promoting HSC activation and ECM deposition during liver fibrogenesis is strongly dependent on GLI1 function. In the human HSC line LX-2, PAX6 binds to the promoter of the GLI1 gene, thereby promoting fibrogenic activities and proliferation. In the same cell line, GLI1 was further shown to be integrated in a complex network of Wnt/β-catenin, which regulates cellular contraction. However, the significance of GLI1 positive perivascular mesenchymal stem-like cells for liver fibrogenesis is still unknown. Publicly available 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 fibrogenesis to generate the proposed large fraction of MFBs.

40Liver fibrosis is potentially reversible. Patients undergoing treatment for HCV infection clearly demonstrate reversal of fibrosis upon complete HCV negativity. However, liver fibrosis is reversible only in the early stages (Fibrosis grades 1 and 2). Once fibrosis crosses a threshold (Fibrosis grades 3 and 4), the fibrogenic type I collagen forms crosslinks and is typically associated with cell damage and inflammation, making it harder to recover. Two events are critical in directing the liver pro-fibrogenic phenotype to recovery - (i) Apoptosis of MFBs in the liver and, (ii) switching of macrophages from a pro-inflammatory to a tissue resolution phenotype. During liver fibrogenesis, the ECM is extensively remodeled leading to accumulation of proteases such as matrix metalloproteinases (MMP) as well as collagenases. However, at the same time, fibrotic liver also accumulates myofibroblast-derived tissue inhibitor of metalloproteinase 1 (TIMP1) which prevents the action of MMPs and ECM turnover. As a result, there is an accumulation of collagen and pro-fibrotic ECM. Over a period of time, accumulation of a large number of crosslinked collagen and elastin fibers lead to sequestering of crosslinked fibers within the tissue beds, making them inaccessible for proteolytic digestion. As the cross links increase, the exposed fibers also become less susceptible to digestion themselves. The hallmark of a recovering fibrotic liver is the termination of cellular injury followed by the absence or disappearance of hepatic MFBs. Studies show that at least 50% of the activated MFBs revert to less fibrogenic or quiescent HSCs. The role of macrophages and the trigger of switching from pro-inflammatory to pro-resolution macrophages during fibrosis is incompletely understood. However, macrophages in the resolving fibrotic liver have been shown to secrete increased levels of MMPs thereby contributing to ECM reorganization. Therefore, polarization of macrophages provides a therapeutic opportunity for the resolution of liver fibrosis.

41Although numerous drugs have beneficial anti-fibrotic effects in vitro and in animal models, none of these drugs has been ultimately shown to be efficacious in the clinic. Moreover, general anti-fibrotic therapies are not available. Instead, clinicians and professional associations have developed some clinical practice guidelines and recommendations for etiology-specific 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 specific etiology of liver injury. As such they typically contain information about disease definition, 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 fibrosis. These include the withdrawal of injurious stimuli, inhibition of ongoing hepatic damage, deactivation and elimination of ECM-producing cells, removal of superfluous scar tissue, counteracting biological mediators driving hepatic inflammation and fibrogenesis, and restoring the normal liver architecture (Figure 12). scRNA-seq and genetic cell tracing experiments have shown that the termination of hepatic fibrosis is associated with a reversal of HSC activation and expression of different inactivation markers. However, reverted HSCs remain in a primed state maintaining a higher responsiveness toward fibrogenic stimuli.

42As outlined, there are many genetic and environmental factors that can cause hepatic fibrosis. Most frequent inherited disorders associated with acute and chronic liver disease include hemochromatosis, Wilson disease, α1-antitrypsin deficiency, and cystic fibrosis. In the case of hemochromatosis, excessive iron can be removed from the body by regularly phlebotomy, or alternatively iron chelating therapy. 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. 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 modifiable risk factors (overweight, tobacco, alcohol, non-steroidal anti-inflammatory drugs) or finally liver transplantation that still remains the sole curative option. In cystic fibrosis resulting of mutations in the gene encoding the cystic fibrosis 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 efficacy is therapy of cystic fibrosis-related liver disease is controversially discussed. In addition, a large number of compounds restoring CFTR protein function (i.e., CFTR modulators) are actually under close investigation.

43In 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 beneficial to lower hepatic inflammation. 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.

44Antiviral treatment strategies are suitable to reduce the 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%. Rigorous infant prophylaxis, early childhood and adult immunization programs as well as vaccination of high-risk individuals significantly contribute to prevalence of HBV transmission worldwide. Established interferon-based therapies are established and new encouraging drugs are currently under clinical evaluation for the treatment of HDV.

45Autoimmune 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). Immunosuppressive treatment consisting of either corticosteroids alone or in combination with the purine analog azathioprine is the recommended first-line medical treatment in autoimmune hepatitis.

46There 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 significant changes in lifestyle and nutrition, in particular weight loss and physical exercise. Nevertheless, there are several drugs currently at various stages of development for the therapy of NASH, possessing anti-inflammatory activity, improve insulin resistance, reduce de novo lipogenesis, modulate lipid transport or oxidation, or evolve anti-apoptotic effects.

47Liver fibrosis 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.

48As discussed, elevated quantities of reactive oxygen species (ROS) are key drivers of hepatic inflammation and fibrosis. 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. 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 profibrogenic mediators, prompt recruitment of circulating inflammatory cells into the liver, and contribute to the activation of HSCs. Therefore, an imbalance between ROS production and degradation play an important role in the pathogenesis of liver fibrosis. Consequently, therapeutic interventions targeting elevated cellular oxidative stress should be beneficial for the treatment of liver fibrosis. In regard to therapy of liver fibrosis, many ROS inhibitors have been tested successfully in pre-clinical animal models. Most of these antioxidants are scavengers that unspecific alleviate ROS accumulation, while others are more selective by targeting defined 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. Most promising are drugs that interfere with the activity of different NADPH oxidase (NOX) subtypes. In experimental models, both the deficiency 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 fibrosis. Likewise, the NOX inhibitor apocynin was therapeutically effective in preventing lipopolysaccharide/d-galactosamine-induced acute liver injury.

49Several cytokines play a crucial role in the pathogenesis of hepatic fibrosis. Commonly, they bind to specific cell-surface exposed receptors, thereby initiating intracellular signaling cascades ending in modified gene expression. Research performed during the last decades has identified a number of different cytokines relevant during the pathogenesis of hepatic fibrosis. Representative cytokines most prominent involved in disease initiation or progression are members belonging to the family of TGF-β, platelet-derived growth factors (PDGF), vascular endothelial growth factors (VEGF), interleukins (IL), fibroblast growth factors (FGF), interferons (IFN), insulin-like growth factors (IGF), TNF-α, epidermal growth factor (EGF), nerve growth factor (NGF), and hepatocyte growth factor (HGF). Their specific activities were comprehensively explored in many in vitro and in vivo models of hepatic fibrosis. However, proposed therapies by interfering with cytokine activities using small interfering RNAs, antisense oligonucleotides, aptamers, soluble receptors, scavenger molecules, therapeutic antibodies, or other biological agents have not been translated to the clinics yet.

50Chemokines are critical immunomodulatory mediators acting in humans through 20 different G-protein-coupled transmembrane receptors. They typically consists of 75–125 amino acids, share a similar tertiary structure, and based on the number and position of cysteine residues can be systematically categorized into four distinct subfamilies, namely CC, CXC, CX3C, and XC followed by the letter L (standing for ‘ligand’) and a consecutive number indicating their temporal isolation. The individual ligands and their cognate receptors (i.e., CCR, CXCR, CX3CR, XCR) form an enormously complex network playing pivotal roles. By far the most studied functions are the control of cell recruitment, inflammation, wound healing, lymphoid trafficking, angiogenesis, and metastasis. For the formation of liver disease, there is now ample evidence that chemokines and their receptors have fundamental importance in both progression and regression of hepatic fibrosis. Therefore, strategies for inhibiting common or individual chemokine activities are presently intensively investigated. Prototypically, the dual specific CC motif chemokine receptor 2/5 (CCR2/CCR5) antagonist cenicriviroc has been experimentally and clinically shown to block fat accumulation, Kupffer cell activation, monocyte recruitment, HSC activation, and fibrosis.

51Besides the elimination of pathogenic causes, usage of replacement therapies, usage of antioxidants, or therapies targeting cytokine or chemokine activities, there are numerous other possibilities to interfere with hepatic fibrosis. In the past, many other drugs or herbal supplements or vitamins were experimentally tested in pre-clinical models of hepatic fibrosis. They act by inducing apoptosis, autophagy or senescence in ECM-producing cells, interfere with pro-fibrogenic target molecules, modulate cell cycle or proliferation synthesis, act hepatoprotective, or generally interfere with gene expression, replication, mitosis or meiosis. However, their efficacy was only successfully proven yet in experimental disease models. Clinical application is hindered in most cases because effective strategies that allow targeting these drugs to fibrogenic effector cells are not available. Other experimental approaches have identified the lysyl oxidase-like 2 (LOXL2) encoding an extracellular copper-dependent amine oxidase catalyzing the covalent cross-linking of collagen and elastin as a promising drug target. In two experimental models of hepatic fibrogenesis, the selective LOXL2/3 inhibitor PXS-5153A was shown to dose-dependently diminish collagen content, thereby reducing disease severity and improve liver function. In line, the preventive treatment with and anti-LOXL2 antibody was able to prevent ongoing experimental hepatic fibrosis. Similarly, targeting Galectin-3 representing a 30 kDa protein with important functions in cell-cell adhesion, cell-matrix interaction, angiogenesis, macrophage activation, inflammation, and collagen synthesis has been identified as a suitable drug candidate.

52Presently, there is much hope that engineered nanoparticles, magnetic-assisted drug delivery techniques, or therapeutic effective transgenes expressed under fibrosis-related promoters can be optimized in the near future to better target individual fibrogenic cell subpopulations.

53Genetic disorders, alcohol abuse, drugs, cholestasis, metabolic disorders, chronic viral hepatitis, parasitic infections and several cryptogenic causes are major causes that provoke liver fibrosis. During this progressive process accumulation of ECM, disruption of the lobular structure, and progressive deterioration of hepatocellular function lead to fatal complications. In particular, the exuberant collagen deposition is one hallmark of fibrogenesis. Work from the last decades have identified a number of different resident and infiltrating cells that can either be activated or transform into a phenotype capable to synthesize ECM. In addition, cell- and animal-based experiments, clinical studies and complex integrated bioinformatics analysis have unraveled soluble mediators, molecular pathways, and pro-fibrogenic genes that are key drivers in the pathogenesis of hepatic disease. However, despite the important progress, there are currently no approved anti-fibrotic drugs that have been ultimately shown to be efficacious in the clinic. Presently, clinical practice guidelines are only etiology-specific. They intend to optimize patient care by withdrawal of injurious stimuli, consumption of antioxidant acting compounds, and lifestyle interventions including healthy food, exercised and controlled weight loss. Nevertheless, many experimental studies and clinical trials are currently being conducted to test drugs targeting more specifically inflammation, the cellular activation process, or the activity of inflammatory or fibrotic-acting cytokines or chemokines. There is hope that these compounds will be of fundamental importance in future treatments aiming at impeding or reversing the fibrogenic process.