**Impact of Glyoxalase-I (Glo-I) and Advanced Glycation End Products (AGEs) in Chronic Liver Disease**

Marcus Hollenbach

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68417

#### **Abstract**

Inflammation caused by oxidative stress (ROS) is a main driver for development of chronic inflammatory liver disease leading to fibrosis and cirrhosis. An important source of ROS constitutes methylglyoxal (MGO). MGO is formed as a by-product in glycolysis, threonine catabolism, and ketone bodies pathway leading to formation of advanced glycation end products (AGEs). AGEs bind to their receptor for AGEs (RAGE) and activate intracellular transcription factors, such as nuclear factor-κB (NF-κB), resulting in production of pro-inflammatory cytokines and ROS. The enzymes glyoxalase-I (Glo-I) and glyoxalase-II (Glo-II) form the glyoxalase system and are essential for the detoxification of methylglyoxal (MGO). This chapter highlights Glo-I and (R)AGE in chronic liver disease with focus on fibrosis and cirrhosis. AGEs and RAGE have been shown to be upregulated in fibrosis, and silencing of RAGE reduced the latter. In contrast, recent study highlighted reduced expression of Glo-I in cirrhosis with consecutive elevation of MGO and oxidative stress. Interestingly, modulation of Glo-I activity by ethyl pyruvate resulted in reduced activation of hepatic stellate cells and reduced fibrosis in CCl<sup>4</sup> model of cirrhosis. In conclusion, Glo-I and R(AGE) are important components in development and progression of chronic liver disease and constitute interesting therapeutic target.

**Keywords:** ethyl pyruvate cirrhosis, fibrosis, methylglyoxal, AGEs

## **1. Introduction**

Oxidate stress (reactive oxygen species, ROS) with consecutive and repetitive inflammation is responsible for development of chronic liver disease. Different etiologies of liver disease lead to damage of hepatocytes, release of pro-inflammatory cytokines, and finally activation

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

of hepatic stellate cells (HSC). Activated HSC transform to myofibroblasts and lead to deposition of collagen, which in turn result in fibrosis and finally cirrhosis. Several molecular mechanisms are involved in this complex interplay, nevertheless the critical step is the activation of HSC by ROS. This chapter focuses on the glyoxalase-I (Glo-I) and related advanced glycation end products (AGEs) with their receptor for AGEs (RAGE) playing an important role in generation and detoxification of ROS. Current knowledge of Glo-I and (R)AGE in chronic liver disease with key aspect to fibrosis and cirrhosis will be highlighted.

## **2. Pathogenesis of fibrosis and cirrhosis**

End-stage liver diseases are mainly caused by viral hepatitis, alcoholism, nonalcoholic fatty liver disease or steatohepatitis (NAFLD/NASH), or rare autoimmune and hereditary disorders. The followed repetitive liver injury caused inflammation, finally resulting in fibrosis and irreversible cirrhosis. Thereby, liver cirrhosis belongs to the global burden of disease responsible for more than one million deaths p.a. [1]. In cirrhosis, altered liver anatomy and reduced liver function are pathognomonic. Development of cirrhosis is characterized by the appearance of regenerative nodules, hepatocyte ballooning, accumulation of fibrotic tissue, disturbed microcirculation, angiogenesis and sinusoidal collapse with defenestration and development of a basement membrane [2]. These alterations of liver architecture lead to reduced liver function and elevation of intrahepatic resistance demonstrated by increased portal pressure with development of ascites and esophageal varices [3, 4]. Nevertheless, portal hypertension is being caused by both structural alterations of liver microarchitecture and hepatic endothelial dysfunction. The latter is characterized by an imbalance of vasoactive components. In fact, there is an hyperresponsiveness and overproduction of vasoconstrictors (mainly endothelin-1 (ET-1)) and an hyporesponsiveness and reduction of vasodilators (mainly nitric oxide (NO)) in the vascular bed of the liver [5–7]. Despite this hypoactive endothelium in hepatic microcirculation, portal hypertension leads to arterial vasodilation, formation of collateral vessels, and hyporesponsiveness to vasoconstrictors due to hyperactive endothelium in splanchnic and systemic circulation with increased NO production. Finally, these alterations result in elevated blood flow to portal vein and a vicious circle of disease [8–11].

The underlying molecular mechanism for development of fibrosis, cirrhosis, and portal hypertension has been intensively investigated over the last decades. Since the liver is formed by parenchymal cells (mainly hepatocytes (HEP)) and nonparenchymal cells (Kupffer cells (KC), hepatic stellate cells (HSC), and liver sinusoidal endothelial cells (LSEC)), both are involved in the development of fibrosis and cirrhosis. Nevertheless, HSC are the main cell type responsible for accumulation of fibrosis and increased intrahepatic vascular resistance. HSC are pericytes surrounding the sinusoids in the space of Disse. HSC are quiescent but became activated upon various stimuli and transform to myofibroblasts [12]. This activation process is a complex interplay between parenchymal and nonparenchymal cells and triggered via inflammatory processes [13]. For instance, deleterious agents (alcohol, LPS) have direct hepatotoxic effects to hepatocytes and trigger the production of reactive oxygen species (ROS). The release of ROS, DNA, and damage-associated molecular pattern (DAMP) leading to activation of KC and innate immune system followed by subsequent production of proinflammatory cytokines such as TNF-α and IL-6 as well as pro-fibrotic factors [14–16]. Also, alcohol consumption increases permeability of the gut resulting in increased levels of portal endotoxins (LPS) with consecutive activation of KC resulting in liver injury and inflammation [17, 18]. Furthermore, inflammation triggers the classical complement pathway activation via C1q [19], followed by production of pro-inflammatory cytokines, and inhibits components of innate immune system. As a consequence of these induced inflammatory processes, activated KC stimulate HSC subsequently leading to fibrosis [20]. This stimulation can result directly by the deleterious agent [21] or via transforming growth factor beta (TGF-β)-dependent mechanisms [22] leading to secretion of TNF-α, IL-6, TIMP-1, MCP-1, collagen-I, and α-SMA [23–25] and finally collagen deposition.

of hepatic stellate cells (HSC). Activated HSC transform to myofibroblasts and lead to deposition of collagen, which in turn result in fibrosis and finally cirrhosis. Several molecular mechanisms are involved in this complex interplay, nevertheless the critical step is the activation of HSC by ROS. This chapter focuses on the glyoxalase-I (Glo-I) and related advanced glycation end products (AGEs) with their receptor for AGEs (RAGE) playing an important role in generation and detoxification of ROS. Current knowledge of Glo-I and (R)AGE in chronic liver

End-stage liver diseases are mainly caused by viral hepatitis, alcoholism, nonalcoholic fatty liver disease or steatohepatitis (NAFLD/NASH), or rare autoimmune and hereditary disorders. The followed repetitive liver injury caused inflammation, finally resulting in fibrosis and irreversible cirrhosis. Thereby, liver cirrhosis belongs to the global burden of disease responsible for more than one million deaths p.a. [1]. In cirrhosis, altered liver anatomy and reduced liver function are pathognomonic. Development of cirrhosis is characterized by the appearance of regenerative nodules, hepatocyte ballooning, accumulation of fibrotic tissue, disturbed microcirculation, angiogenesis and sinusoidal collapse with defenestration and development of a basement membrane [2]. These alterations of liver architecture lead to reduced liver function and elevation of intrahepatic resistance demonstrated by increased portal pressure with development of ascites and esophageal varices [3, 4]. Nevertheless, portal hypertension is being caused by both structural alterations of liver microarchitecture and hepatic endothelial dysfunction. The latter is characterized by an imbalance of vasoactive components. In fact, there is an hyperresponsiveness and overproduction of vasoconstrictors (mainly endothelin-1 (ET-1)) and an hyporesponsiveness and reduction of vasodilators (mainly nitric oxide (NO)) in the vascular bed of the liver [5–7]. Despite this hypoactive endothelium in hepatic microcirculation, portal hypertension leads to arterial vasodilation, formation of collateral vessels, and hyporesponsiveness to vasoconstrictors due to hyperactive endothelium in splanchnic and systemic circulation with increased NO production. Finally, these alterations result in

disease with key aspect to fibrosis and cirrhosis will be highlighted.

elevated blood flow to portal vein and a vicious circle of disease [8–11].

The underlying molecular mechanism for development of fibrosis, cirrhosis, and portal hypertension has been intensively investigated over the last decades. Since the liver is formed by parenchymal cells (mainly hepatocytes (HEP)) and nonparenchymal cells (Kupffer cells (KC), hepatic stellate cells (HSC), and liver sinusoidal endothelial cells (LSEC)), both are involved in the development of fibrosis and cirrhosis. Nevertheless, HSC are the main cell type responsible for accumulation of fibrosis and increased intrahepatic vascular resistance. HSC are pericytes surrounding the sinusoids in the space of Disse. HSC are quiescent but became activated upon various stimuli and transform to myofibroblasts [12]. This activation process is a complex interplay between parenchymal and nonparenchymal cells and triggered via inflammatory processes [13]. For instance, deleterious agents (alcohol, LPS) have direct hepatotoxic effects to hepatocytes and trigger the production of reactive oxygen species (ROS). The release of ROS, DNA, and damage-associated molecular pattern (DAMP) leading

**2. Pathogenesis of fibrosis and cirrhosis**

212 Liver Cirrhosis - Update and Current Challenges

As mentioned above, pro-inflammatory factors (TNF-α, IL-1β, IL-6) are also involved in the activation of HSC. In this regard, activation of the transcription factor nuclear factor-κB (NFκB) and subsequent overexpression of pro-inflammatory cytokines are important pathways. NF-κB, thereby, is activated by growth factors, cytokines, bacterial and viral factors, and ROS and regulates by itself pro-inflammatory cytokines (like COX-2 or IL-6) [26, 27].

Beside the production of collagen and accumulation of fibrotic tissue, HSC are involved in increased intrahepatic vascular resistance not only via structural changes. Transformation of HSC to myofibroblasts was accompanied by stimulation of rho kinase leading to activation of contractile filaments of HSC and subsequently vasoconstriction of sinusoids [28].

Another key player in the development of fibrosis comprises LSEC. They form the first line of defense protecting the liver from injury. Inflammation by LPS or ROS resulted in dysfunction of LSEC [29] indicated by disturbed sinusoidal microcirculation, defenestration, hypoxia, and pathological angiogenesis [30]. In contrast, both direct deterioration of LSEC and vasoconstriction of HSC result in impaired release of vasodilators from LSEC leading to a vicious circle of disease. In this regard, disturbed regulation of NO production in cirrhosis depends on activity of endothelial NO synthase (eNOS) and increased degradation due to phosphodiesterases, that is, PDE-5 [31]. Although eNOS expression is upregulated in sinusoidal area in cirrhosis, eNOS activity has been shown to be reduced by caveolin-eNOS binding [32] and was diminished by several post-translational modifications of the endothelial NO synthase (eNOS) [9]. In contrast, in splanchnic circulation, eNOS is upregulated [9] with increased enzyme activity in portal hypertension and regulated by phosphorylation of protein kinase B (Akt) [33]. Beside upregulation of eNOS, production of NO is also related to induction of the inducible form of the NO synthase, iNOS. iNOS is mainly stimulated by the presence of endotoxin and pro-inflammatory cytokines, all of whom occur in development of cirrhosis [34]. Indeed, recent study showed stimulation of iNOS rather than eNOS in splanchnic circulation by LPS, indicating an important role of iNOS in portal hypertension after bacterial translocation to mesenteric vessels [35]. Finally, all these alterations result in a hyperdynamic circulation with elevated blood flow to portal vein and further increase of portal pressure [8–10].

In conclusion, cirrhosis demonstrates the end stage of liver disease with disturbed liver architecture and impaired liver function. Generation of ROS and stimulation of various inflammatory pathways are critical steps in activation of HSC as the main driver for fibrosis. Despite these findings, the use of antioxidants (vitamin E, N-acetylcysteine, coenzyme Q, and others) in patients with alcoholic liver disease has failed to show an efficacy in improving disease conditions [36–38].

## **3. Glyoxalase system and R(AGE)**

An important role in regulation and formation of ROS and oxidative stress comprises the glyoxalase system. This enzymatic system was first discovered in 1913 [39] and constitutes two cytosolic enzymes, glyoxalase-I (Glo-I, EC 4.4.1.5) and glyoxalase-II (Glo-II, EC 3.1.2.6.). Glo-I is responsible for the catalytic conversion of α-oxo aldehydes, for instance, methylglyoxal (MGO), into the hemithioacetal S-D-Lactoylglutathione using L-glutathione (GSH) as a cofactor. Further substrates of Glo-I are hydroxypyruvaldehyde, hydroxypyruvate aldehyde phosphate, glyoxal, phenylglyoxal, 4,5-dioxovalerate, alkyl and arylglyoxales [40–43]. Glo-II hydrolyzes the reaction of S-D-Lactoylglutathione to H<sup>2</sup> O and D-lactate with regeneration of GSH (**Figure 1**). Thereby, Glo-I demonstrates the rate limiting step [42, 44], and Glo-II is of subordinate interest in inflammatory research.

MGO is the main substrate of Glo-I [45] and has been described as a reactive carbonyl compound that is formed as a by-product in glycolysis [46], ketone body metabolism, and threonine catabolism [47–49]. MGO leads to cell cytotoxicity in high concentrations through

**Figure 1.** Glyoxalase system. Glyoxalase-I and glyoxalase-II comprise the glyoxalase system for detoxification of MGO. Glutathione is necessary as cofactor and is regenerated by Glo-II. Adapted from [43].

reaction with nucleotides, phospholipids, and proteins [50, 51], resulting in the formation of "advanced glycation end products (AGEs)" and reactive oxygen species (ROS) via AGEs or non-enzymatic reaction with hydrogen peroxide [52]. In this regard, MGO has shown to be involved in various inflammatory processes such as diabetes, aging, renal insufficiency, hypertension, or cancer [60–64].

these findings, the use of antioxidants (vitamin E, N-acetylcysteine, coenzyme Q, and others) in patients with alcoholic liver disease has failed to show an efficacy in improving disease

An important role in regulation and formation of ROS and oxidative stress comprises the glyoxalase system. This enzymatic system was first discovered in 1913 [39] and constitutes two cytosolic enzymes, glyoxalase-I (Glo-I, EC 4.4.1.5) and glyoxalase-II (Glo-II, EC 3.1.2.6.). Glo-I is responsible for the catalytic conversion of α-oxo aldehydes, for instance, methylglyoxal (MGO), into the hemithioacetal S-D-Lactoylglutathione using L-glutathione (GSH) as a cofactor. Further substrates of Glo-I are hydroxypyruvaldehyde, hydroxypyruvate aldehyde phosphate, glyoxal, phenylglyoxal, 4,5-dioxovalerate, alkyl and arylglyoxales [40–43]. Glo-II

GSH (**Figure 1**). Thereby, Glo-I demonstrates the rate limiting step [42, 44], and Glo-II is of

MGO is the main substrate of Glo-I [45] and has been described as a reactive carbonyl compound that is formed as a by-product in glycolysis [46], ketone body metabolism, and threonine catabolism [47–49]. MGO leads to cell cytotoxicity in high concentrations through

**Figure 1.** Glyoxalase system. Glyoxalase-I and glyoxalase-II comprise the glyoxalase system for detoxification of MGO.

Glutathione is necessary as cofactor and is regenerated by Glo-II. Adapted from [43].

O and D-lactate with regeneration of

conditions [36–38].

214 Liver Cirrhosis - Update and Current Challenges

**3. Glyoxalase system and R(AGE)**

hydrolyzes the reaction of S-D-Lactoylglutathione to H<sup>2</sup>

subordinate interest in inflammatory research.

Important MGO-derived AGEs are the non-fluorescent products 5-hydro-5-methylimidazolone (MG-H1) and tetrahydropyrimidine (THP) as well as the major fluorescent product, argpyrimidine [53, 54]. Other non-MGO-derived AGEs comprise Nε -carboxymethyllysine (CML), pyrraline, or pentosidine [55]. The effects of AGEs have been allocated to their antagonistic receptor systems. The receptor for AGEs (RAGE) mediates generation of ROS, inflammation, angiogenesis, and proliferation [56, 57]. In contrast, AGE receptors (AGE-Rs), for instance, AGE-R1, are responsible for detoxification and clearance of AGEs [58]. Upon binding of AGEs to RAGE, various signal transduction pathways are activated. Recent studies showed involvement of the extracellular signal-regulated kinase 1/2 (ERK1/2), phosphoinositide 3-kinase (PI3-K)/protein kinase B (AKT), Janus kinase 2 (JAK2), and Rho GTPases, finally resulting in activation of NF-κB and production of pro-inflammatory cytokines (**Figure 3**) [59]. In addition, stimulation of RAGE resulted in activation of transforming growth factor (TGF-β) pathway and induced vascular endothelial growth factor (VEGF) overexpression [57].

In the last years, structure and genomic sequence of Glo-I was intensively analyzed. Glo-I is a dimer and consists in mammalian of two identical subunits with a molecular mass of 43–48 kDa [60]. Each subunit contains a zinc ion in its active center, whereas the apoenzyme remains catalytically inactive [45, 61]. The active center of Glo-I is localized between both monomers and comprises two structurally equivalent residues from each domain (Gln-33A, Glu-99A, His-126B, Glu-172B) and two water molecules indicating an octahedral arrangement [54, 62]. The protein sequence of Glo-I consists of 184 amino acids with post-translational modification of N-terminal Met [62].

Genomic analysis revealed three distinct phenotypes of Glo-I: GLO 1-1, GLO 1-2, and GLO 2-2 representing homo- and heterozygous expression of *GLO1* und *GLO2* [63, 64]. Gene locus of Glo-I is determined on chromosome six between centromere and human leukocyte antigen (HLA)-DR gene [65, 66]. Demographic studies showed higher distribution of *GLO1* in Alaska and lower *GLO1* allocation in southern and eastern Europe, America, Africa, and India [67].

Genetic sequencing identified association of distinct Glo-I phenotypes and Glo-I SNPs with diabetes [68], cardiovascular diseases [69], schizophrenia [70], autism [71, 72], anxiety [73], and cancer [74, 75]. These findings led to preliminary anti-tumor effects of Glo-I inhibition by siRNA or enzymatic inhibition in different cancer models [76–79]. In this regard, wellstudied Glo-I inhibitors are S-ρ-bromobenzylglutathione or S-ρ-bromobenzyl-glutathione cyclopentyl diester [77, 80], methotrexate [81], indomethacin [82], troglitazone [83], and flavonoids [84, 85] showing anti-inflammatory and anti-tumor effects. Furthermore, an Glo-I inducer led to improved glycemic control and vascular function in 29 obese patients [86].

In a nutshell, Glo-I is responsible for detoxification of MGO and prevention of MGO-related formation of AGEs and ROS. Therefore, Glo-I and (R)AGE are involved in different pathophysiological inflammatory processes.

## **4. Glo-I and R(AGE) in fibrosis, cirrhosis, and NAFLD/NASH**

#### **4.1. Glo-I**

To date, although Glo-I revealed an important role in inflammation, data about Glo-I in chronic liver disease remain preliminary. In an experimental approach of CCl<sup>4</sup> -induced cirrhosis, Glo-I was analyzed *in vivo* and *in vitro* [87]. Wistar rats were treated with inhalative CCl<sup>4</sup> three times a week to induce early cirrhosis (without ascites) after 8 weeks or advanced cirrhosis (with ascites) after 12 weeks. Furthermore, primary liver cells from cirrhotic and noncirrhotic livers were isolated via portal vein perfusion and analysis of Glo-I was performed. Glo-I could be detected in HEP, HSC, and LSEC with highest expression on protein and mRNA levels in HEP. Furthermore, Glo-I expression was reduced in early and advanced cirrhosis in both whole liver and primary liver cells (**Figure 2A**). The reduction in Glo-I expression was greater with increasing severity of liver disease. Interestingly, the reduction of Glo-I was accompanied by an increase of MGO in cirrhosis (**Figure 2B**). This accumulation of MGO would lead to increased formation of AGEs and finally augment oxidative stress with ongoing inflammation in chronic liver disease [87]. So far, the reduction of Glo-I with consecutive increase of MGO would provide an explanation for perpetuating liver inflammation in advanced stages of liver disease.

Furthermore, modulation of Glo-I activity with the anti-inflammatory drug ethyl pyruvate (EP) was performed to analyze impact of Glo-I in initiation and progression of cirrhosis. EP is an α-oxo-carbonic acid and ester of pyruvate. EP came in focus due to anti-inflammatory effects of pyruvate but low stability in aqueous solution [88]. Therefore, EP constitutes a more stable compound and exerts anti-inflammatory and protective effects in a lot of ROS-mediated models [89, 90]. Therefore, a possible molecular basis for the anti-inflammatory effects of EP was assumed to be the inhibition of specific Glo-I activity [91].

Since EP showed protective effects in acute liver failure [92–95] and development of fatty liver [96], effect of EP on activation of HSC, as it might occur in initial stadium of cirrhosis, was analyzed. Stimulation of HSC with LPS for 24 hours led to increased levels of α-SMA, indicating activation of HSC and production of collagen deposit. This stimulation could be abrogated by modulation of Glo-I activity by means of EP (**Figure 2c**). Underlying mechanisms involve stimulation of Nrf2 as well as reduction of NF-κB and ERK/pERK by EP. Additional *in vivo* experiments revealed reduced collagen deposit in Wistar rats that were treated with CCl<sup>4</sup> for 12 weeks and i.p. EP [87]. Furthermore, EP-treated rats revealed significantly less Sirius red staining and consequently less fibrosis compared with controls receiving saline (**Figure 2D**).

Indeed, anti-inflammatory treatment of several diseases with EP might be a promising future clinical approach. However, EP was analyzed in a clinical trial (phase-II multicenter doubleblind placebo-controlled study) in high-risk patients undergoing cardiac surgery with cardiopulmonary bypass. This trial was performed in 13 US hospitals including patients with Impact of Glyoxalase-I (Glo-I) and Advanced Glycation End Products (AGEs) in Chronic Liver Disease http://dx.doi.org/10.5772/intechopen.68417 217

**Figure 2.** Glyoxalase-I in CCl<sup>4</sup> -induced cirrhosis. **(A)**, Glo-I expression was reduced in early (8 week CCl<sup>4</sup> -treatment) and advanced (12 week CCl<sup>4</sup> -treatment) cirrhosis in Western blot. Wistar rats were treated three times per week with inhalative CCl<sup>4</sup> for induction of cirrhosis. **(B)**, MGO levels were significantly elevated in cirrhosis, indicated by ELISA-analysis. **(C)**, treatment of stellate cells (HSC) for 24 hours with LPS revealed increased production of α-SMA. Cotreatment with Glo-I modulator ethyl pyruvate (EP) abolished the LPS-induced effects. **(D)**, Wistar rats were treated with CCl<sup>4</sup> and i.p. EP or saline from week 8 to 12. Sirius red staining indicated significantly less fibrosis in EP-treated animals. \* p < 0.05, \*\* p < 0.01, \*\*\* p < 0.001. Adapted from [87].

a Parsonnet risk score > 15 undergoing coronary artery bypass graft and/or cardiac valvular surgery with cardiopulmonary bypass. 102 subjects received either placebo (53) or 7.500 mg (90 mg/kg) EP (49) intravenously followed by five more doses every 6 hours. The primary endpoint was a combination of death, prolonged mechanical ventilation, renal failure, or need of vasoconstrictors. No statistically significant differences were observed between groups with regard to clinical parameters or markers of systemic inflammation [97]. Despite these disappointing results in the first clinical trial, it should be kept in mind that underlying molecular mechanisms in cardiac surgery with cardiopulmonary bypass are complex and at least partly different from ROS models showing protective effects of EP. Another clinical study design, for example, liver fibrosis, pancreatitis, septic shock, might be more promising for this interesting agent.

In summary, targeting Glo-I with EP in cirrhosis revealed an innovative therapeutic target. Nevertheless, further research needs to confirm the aforementioned results in further animal experiments and clinical trials.

#### **4.2. AGEs**

In a nutshell, Glo-I is responsible for detoxification of MGO and prevention of MGO-related formation of AGEs and ROS. Therefore, Glo-I and (R)AGE are involved in different patho-

To date, although Glo-I revealed an important role in inflammation, data about Glo-I in chronic

a week to induce early cirrhosis (without ascites) after 8 weeks or advanced cirrhosis (with ascites) after 12 weeks. Furthermore, primary liver cells from cirrhotic and noncirrhotic livers were isolated via portal vein perfusion and analysis of Glo-I was performed. Glo-I could be detected in HEP, HSC, and LSEC with highest expression on protein and mRNA levels in HEP. Furthermore, Glo-I expression was reduced in early and advanced cirrhosis in both whole liver and primary liver cells (**Figure 2A**). The reduction in Glo-I expression was greater with increasing severity of liver disease. Interestingly, the reduction of Glo-I was accompanied by an increase of MGO in cirrhosis (**Figure 2B**). This accumulation of MGO would lead to increased formation of AGEs and finally augment oxidative stress with ongoing inflammation in chronic liver disease [87]. So far, the reduction of Glo-I with consecutive increase of MGO would provide an explanation for perpetuating liver inflammation in advanced stages of liver disease.

Furthermore, modulation of Glo-I activity with the anti-inflammatory drug ethyl pyruvate (EP) was performed to analyze impact of Glo-I in initiation and progression of cirrhosis. EP is an α-oxo-carbonic acid and ester of pyruvate. EP came in focus due to anti-inflammatory effects of pyruvate but low stability in aqueous solution [88]. Therefore, EP constitutes a more stable compound and exerts anti-inflammatory and protective effects in a lot of ROS-mediated models [89, 90]. Therefore, a possible molecular basis for the anti-inflammatory effects of EP

Since EP showed protective effects in acute liver failure [92–95] and development of fatty liver [96], effect of EP on activation of HSC, as it might occur in initial stadium of cirrhosis, was analyzed. Stimulation of HSC with LPS for 24 hours led to increased levels of α-SMA, indicating activation of HSC and production of collagen deposit. This stimulation could be abrogated by modulation of Glo-I activity by means of EP (**Figure 2c**). Underlying mechanisms involve stimulation of Nrf2 as well as reduction of NF-κB and ERK/pERK by EP. Additional *in vivo* experiments revealed reduced collagen deposit in Wistar rats that were treated with CCl<sup>4</sup>

12 weeks and i.p. EP [87]. Furthermore, EP-treated rats revealed significantly less Sirius red staining and consequently less fibrosis compared with controls receiving saline (**Figure 2D**). Indeed, anti-inflammatory treatment of several diseases with EP might be a promising future clinical approach. However, EP was analyzed in a clinical trial (phase-II multicenter doubleblind placebo-controlled study) in high-risk patients undergoing cardiac surgery with cardiopulmonary bypass. This trial was performed in 13 US hospitals including patients with


three times

for

**4. Glo-I and R(AGE) in fibrosis, cirrhosis, and NAFLD/NASH**

was analyzed *in vivo* and *in vitro* [87]. Wistar rats were treated with inhalative CCl<sup>4</sup>

liver disease remain preliminary. In an experimental approach of CCl<sup>4</sup>

was assumed to be the inhibition of specific Glo-I activity [91].

physiological inflammatory processes.

216 Liver Cirrhosis - Update and Current Challenges

**4.1. Glo-I**

In contrast to straightforward evidence of Glo-I in chronic liver disease, several groups analyzed AGEs in liver fibrosis, cirrhosis, and NASH. In cirrhotic patients, limited amount of methylglyoxal-modified proteins were found to be elevated compared to controls [98]. Another study revealed increased levels of CML-AGEs in blood plasma of cirrhotic patients. Also, CML levels correlated with severity of disease [99]. Additional studies confirmed the observations of increased CML levels in fibrosis and cirrhosis [100, 101]. These clinical findings were supported by laboratory analysis: *in vitro* treatment of HSC with AGEs resulted in enhanced production of oxidative stress providing evidence of AGEs-involvement in fibrosis [102]. Conversely, oxidative stress was found to elevate levels of CML in rats [103] and incubation of HSC with AGEs led to elevation of α-SMA, TGF-β, and collagen-I [104]. In addition, treatment of rat hepatocyte cultures with AGEs reduced cell viability [105]. In an interesting translational study, CML-AGEs were positively correlated with liver stiffness in patients with chronic hepatitis C. *In vitro* data showed in this study enhanced cell proliferation of HSC treated with BSA-AGEs (CML) and increased production of α-SMA. In contrast, in another study, intraperitoneal administration of AGE-rat serum albumin (CML) revealed increased levels of α-SMA and fibrosis in a model of bile duct ligation [106]. Furthermore, AGEs were found to induce autophagy which subsequently contributes to the fibrosis in patients with chronic hepatitis C [107]. The finding that AGEs were elevated in fibrosis and treatment with AGEs-induced fibrosis led to an interventional approach targeting AGEs to prevent induction of chronic liver disease. Indeed, inhibition of CML resulted in attenuation of CML-induced levels of α-SMA and ROS in HSC [108].

Another model to study fibrosis belongs to metabolic liver diseases: induction of NASH by means of methionine choline deficient diet (MCD). Therefore, hepatic steatosis induced by MCD showed accumulation of CML, and CML was associated with grade of hepatic inflammation and gene expression of inflammatory markers (PAI-1, IL-8, and CRP) [109]. AGEs have also been shown to be involved in etiology of insulin resistance and diabetes [110], and rats fed with a diet rich in AGEs showed elevated oxidative stress and hepatic inflammation leading to NASH [111]. In addition, high dietary AGEs increased hepatic AGEs levels and induced liver injury, inflammation, and liver fibrosis via oxidative stress in activated HSC [112]. Another interesting study investigated the underlying mechanism of AGEs-crosstalk in NASH. AGEs induced NOX2 leading to downregulation of Sirt1/Timp3 and finally resulting in activation of TNF-α converting enzyme and inflammation. These pro-inflammatory cascades finally led to NASH and fibrosis [113]. Interventional studies on AGEs reduction in NASH also revealed promising results. The flavonoid curcumin eliminated the inflammatory effects of AGEs in HSC by interrupting leptin signaling and activating transcription factor Nrf2, which led to the elevation of cellular glutathione levels and the attenuation of oxidative stress [114]. In addition, curcumin decreased activation and proliferation of HSC by AGEs and induced gene expression of AGE-clearing receptor AGE-R1 [115]. The use of the LDL-lowering drug atorvastatin [116] or combination therapy of telmisartan and nateglinide [117] also decreased levels of AGEs in patients with NASH and dyslipidemia, leading to improvement of steatosis, nonalcoholic fatty liver disease activity score, and amelioration of insulin resistance. Another study evaluated effects of aqueous extracts from Solanum nigrum (AESN). AESN could reduce the AGE-induced expression of collagen-II, MMP-2, and α-SMA in HSC. Also, AESN improved insulin resistance and hyperinsulinemia and downregulated lipogenesis, finally preventing fibrosis [118].

Having the auspicious and conclusive effects of AGEs-lowering drugs in fibrosis in mind, it should be noted that mainly CML-AGEs were investigated. Therefore, it should be considered that CML-AGEs are rarely produced via reaction of MGO but are rather formed in lipoxidation and glycoxidation independent of MGO [119].

#### **4.3. RAGE**

Another study revealed increased levels of CML-AGEs in blood plasma of cirrhotic patients. Also, CML levels correlated with severity of disease [99]. Additional studies confirmed the observations of increased CML levels in fibrosis and cirrhosis [100, 101]. These clinical findings were supported by laboratory analysis: *in vitro* treatment of HSC with AGEs resulted in enhanced production of oxidative stress providing evidence of AGEs-involvement in fibrosis [102]. Conversely, oxidative stress was found to elevate levels of CML in rats [103] and incubation of HSC with AGEs led to elevation of α-SMA, TGF-β, and collagen-I [104]. In addition, treatment of rat hepatocyte cultures with AGEs reduced cell viability [105]. In an interesting translational study, CML-AGEs were positively correlated with liver stiffness in patients with chronic hepatitis C. *In vitro* data showed in this study enhanced cell proliferation of HSC treated with BSA-AGEs (CML) and increased production of α-SMA. In contrast, in another study, intraperitoneal administration of AGE-rat serum albumin (CML) revealed increased levels of α-SMA and fibrosis in a model of bile duct ligation [106]. Furthermore, AGEs were found to induce autophagy which subsequently contributes to the fibrosis in patients with chronic hepatitis C [107]. The finding that AGEs were elevated in fibrosis and treatment with AGEs-induced fibrosis led to an interventional approach targeting AGEs to prevent induction of chronic liver disease. Indeed, inhibition of CML resulted in attenuation of CML-induced

Another model to study fibrosis belongs to metabolic liver diseases: induction of NASH by means of methionine choline deficient diet (MCD). Therefore, hepatic steatosis induced by MCD showed accumulation of CML, and CML was associated with grade of hepatic inflammation and gene expression of inflammatory markers (PAI-1, IL-8, and CRP) [109]. AGEs have also been shown to be involved in etiology of insulin resistance and diabetes [110], and rats fed with a diet rich in AGEs showed elevated oxidative stress and hepatic inflammation leading to NASH [111]. In addition, high dietary AGEs increased hepatic AGEs levels and induced liver injury, inflammation, and liver fibrosis via oxidative stress in activated HSC [112]. Another interesting study investigated the underlying mechanism of AGEs-crosstalk in NASH. AGEs induced NOX2 leading to downregulation of Sirt1/Timp3 and finally resulting in activation of TNF-α converting enzyme and inflammation. These pro-inflammatory cascades finally led to NASH and fibrosis [113]. Interventional studies on AGEs reduction in NASH also revealed promising results. The flavonoid curcumin eliminated the inflammatory effects of AGEs in HSC by interrupting leptin signaling and activating transcription factor Nrf2, which led to the elevation of cellular glutathione levels and the attenuation of oxidative stress [114]. In addition, curcumin decreased activation and proliferation of HSC by AGEs and induced gene expression of AGE-clearing receptor AGE-R1 [115]. The use of the LDL-lowering drug atorvastatin [116] or combination therapy of telmisartan and nateglinide [117] also decreased levels of AGEs in patients with NASH and dyslipidemia, leading to improvement of steatosis, nonalcoholic fatty liver disease activity score, and amelioration of insulin resistance. Another study evaluated effects of aqueous extracts from Solanum nigrum (AESN). AESN could reduce the AGE-induced expression of collagen-II, MMP-2, and α-SMA in HSC. Also, AESN improved insulin resistance and hyperinsulinemia and downregulated

Having the auspicious and conclusive effects of AGEs-lowering drugs in fibrosis in mind, it should be noted that mainly CML-AGEs were investigated. Therefore, it should be considered

levels of α-SMA and ROS in HSC [108].

218 Liver Cirrhosis - Update and Current Challenges

lipogenesis, finally preventing fibrosis [118].

The pattern recognition receptor RAGE belongs to the immunoglobulin superfamily with a molecular mass of 47–55 kDa. RAGE expression is stimulated under inflammatory conditions such as diabetes, cardiovascular diseases, or cancer [120]. RAGE has been shown to be activated by MGO- and non-MGO-derived AGEs as well as multiple ligands. Binding to RAGE results in activation of transcription factors, such as NF-κB [121], leading to the release of pro-inflammatory cytokines.

Indeed, several studies revealed participation of RAGE in fibrosis: Upon stimulation with AGE-rat serum albumin containing mainly CML, levels of RAGE, α-SMA, hydroxyproline, and Sirius red were elevated in a fibrosis model of bile duct ligation (BDL) [106, 122]. Interestingly, RAGE was found to be predominantly expressed in HSC. RAGE was stimulated in HSC during transformation to myofibroblasts, and RAGE was colocalized with α-SMA and induced by TGF-β. In addition, RAGE was expressed in filopodial membranes of myofibroblasts suggesting a role of RAGE in spreading and migration of activated HSC in fibrogenesis [123]. Further analysis provided evidence for crosstalk of RAGE and TGF-β: AGEs-induced upregulation of RAGE induced TGF-β, TNF-α, and IL-8. Interestingly, RAGE also stimulated anti-inflammatory cytokines IL-2 and IL-4 indicating a negative feedback mechanism and inhibitory crosstalk between TGF-β and RAGE [124]. In the next step, effect of RAGE inhibition on inflammation and fibrosis was discovered. First, curcumin was found to reduce, besides its AGEs-lowering effects, the gene expression of RAGE via elevation of PPAR-γ [125]. Furthermore, RAGE expression was diminished by means of RAGE siRNA in primary rat HSC resulting in downregulation of IL-6, TNF-α, and TGF-β [126]. In a following *in vivo* study, effects of repetitive RAGE siRNA in an olive oil model of fibrosis were analyzed. RAGE siRNA was injected twice weekly in the tail vein of Sprague-Dawley rats. After 6 weeks, reduced expressions of RAGE, TNF-α, IL-6, extracellular matrix, hyaluronic acid, and procollagen III were found. Also, activation of HSC and NF-κB was reduced in siRNA-treated animals attenuating the initiation and progression of fibrosis [127]. Additional studies revealed protective effects of anti-RAGE antibodies in BDL-induced acute liver injury [128, 129].

Growing evidence for implication of RAGE in fibrosis was found in NASH. Methionine choline deficient (MCD) diet caused steatosis and increased RAGE, inflammation, and fibrosis [112]. Recently, fatty acids stimulated CML accumulation and subsequently elicited RAGE induction [109]. Another group found upregulation of RAGE in the liver of aged mice with consecutive elevated oxidative stress shown by analysis of malondialdehyde. Blocking of RAGE by anti-RAGE-antibody revealed in this study prolonged survival of animals [130].

In a nutshell, various studies confirmed implication of Glo-I and (R)AGE in inflammatory liver disease and fibrosis. Especially targeting Glo-I in cirrhosis highlighted the meaning of MGO-induced liver damage and offers new therapeutic opportunities. Nevertheless, further research in this topic will uncover the exact role of Glo-I in chronic liver disease and possible translation to clinical approach (see **Figure 3**).

**Figure 3.** Impact of Glo-I and (R)AGE in cirrhosis. MGO reacts with proteins, nucleotides, and lipids leading to formation of AGEs. AGEs bind to RAGE and activate several signal pathways (including MAPK (ERK1/2, p38, JNK), PI3-K/AKT, and JAK2/STAT1), finally leading to activation of NF-κB. In a consequence, the induced production of TGF-β and proinflammatory cytokines activate quiescent stellate cells. HSC transform to myofibroblasts and produce pro-fibrotic factors and collagen. The collagen deposition in the liver will lead to fibrosis and finally cirrhosis. Reduction of Glo-I will perpetuate both, initiation and progression of cirrhosis due to increase of MGO and a vicious circle of disease. MGO: methylglyoxal, AGEs: advanced glycation end products, RAGE: receptor for advanced glycation end products, Glo-I: glyoxalase-I, HSC: hepatic stellate cells, MAPK: mitogen-activated protein kinase, PI3-K: phosphoinositide 3-kinase, AKT: protein kinase B, JAK2: Janus kinase 2, STAT1: signal transducer and activator of transcription-1, JNK: c-Jun N-terminal kinase, and NF-κB: nuclear factor-κB.

## **Abbreviations**



## **Author details**

Marcus Hollenbach

Address all correspondence to: marcus.hollenbach@web.de

Department of Medicine, Neurology, and Dermatology, Division of Gastroenterology and Rheumatology, University of Leipzig, Leipzig, Germany

## **References**

**Abbreviations**

N-terminal kinase, and NF-κB: nuclear factor-κB.

220 Liver Cirrhosis - Update and Current Challenges

AKT protein kinase B EP ethyl pyruvate ET-1 endothelin-1 Glo-I glyoxalase-I Glo-II glyoxalase-II GSH L-glutathione

HCC hepatocellular carcinoma

AGEs advanced glycation end products

**Figure 3.** Impact of Glo-I and (R)AGE in cirrhosis. MGO reacts with proteins, nucleotides, and lipids leading to formation of AGEs. AGEs bind to RAGE and activate several signal pathways (including MAPK (ERK1/2, p38, JNK), PI3-K/AKT, and JAK2/STAT1), finally leading to activation of NF-κB. In a consequence, the induced production of TGF-β and proinflammatory cytokines activate quiescent stellate cells. HSC transform to myofibroblasts and produce pro-fibrotic factors and collagen. The collagen deposition in the liver will lead to fibrosis and finally cirrhosis. Reduction of Glo-I will perpetuate both, initiation and progression of cirrhosis due to increase of MGO and a vicious circle of disease. MGO: methylglyoxal, AGEs: advanced glycation end products, RAGE: receptor for advanced glycation end products, Glo-I: glyoxalase-I, HSC: hepatic stellate cells, MAPK: mitogen-activated protein kinase, PI3-K: phosphoinositide 3-kinase, AKT: protein kinase B, JAK2: Janus kinase 2, STAT1: signal transducer and activator of transcription-1, JNK: c-Jun


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## **Regenerative Medicine in Liver Cirrhosis: Promises and Pitfalls**

Asima Tayyeb, Fareeha Azam, Rabia Nisar, Rabia Nawaz, Uzma Qaisar and Gibran Ali

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68729

#### **Abstract**

Liver cirrhosis is irreversible and mostly ends up with complete loss of liver function/ end‐stage liver failure, and the only proven treatment is liver transplantation. Scarcity of donor, high cost, lifelong immunosuppression, and surgical complications are the major issues associated with liver transplantation and these urge to look for alternate therapeu‐ tic approaches. Advancements in the field of regenerative medicine are arising hope for the treatment of liver cirrhosis. This chapter deals with the scope of liver regenerative medicine in the treatment of liver cirrhosis. Review of the literature showed that liver regenerative medicine no doubt holds great promises and added a lot of hope to the cure of liver diseases. Primarily, cell‐based therapies had shown great potential to treat liver cirrhosis. Successful clinical human trials further strengthen their significance in the field. However, recent trends in liver regenerative medicine are focusing on the development of tissue engineering leading to generation of the whole organ. Despite advantages, liver regenerative medicine has several limitations and sometimes been over‐optimistically interpreted. In conclusion, the current scenario advocates to conduct more preclinical and clinical trials to effectively replace liver transplantation with liver regenerative medi‐ cine to treat liver diseases.

**Keywords:** regenerative medicine, stem cells, hepatocytes, tissue engineering

## **1. Introduction**

Liver is one of the largest and most important metabolic organs in the human body with considerable regeneration capacity. However, in prolonged hepatic injuries, the regeneration capacity of hepatocytes times out and a cascade of life‐threatening complications is initiated

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

leading to liver cirrhosis. Liver cirrhosis is irreversible and mostly ends up with complete loss of liver function/end‐stage liver failure. End‐stage liver failure with high rates of morbid‐ ity and mortality poses a significant threat to human health as well as economy throughout the world [1]. As current pharmacological treatments are inefficient to reverse this loss, liver transplantation is the only effective lifesaving option. Since the first liver transplantation in 1963, the number of cases requiring transplantation are considerably increasing with the pas‐ sage of time. Despite the success of liver transplantation, there is a gap between demand and supply. Only 30–50% of annual liver donation desires are fulfilled and at least about 15% patients die while being on the waiting list [2, 3]. Besides scarcity of liver donors, high cost, postoperative graft rejection, and long‐term immune‐suppression are few more serious constraints associated with liver transplant [4]. Therefore, it is crucial to look for effective and operative alternate approaches of liver transplantation.

Advancements in the field of regenerative medicine open up new horizons and arising hope in the treatment of irreversibly damaged liver cirrhosis. Liver regenerative medicine mainly emphasizes on the establishment of new therapies to either functionally restore the chronically damaged liver tissue or to develop the entire new organ [5]. Elucidation of cel‐ lular and molecular mechanisms during the last couple of decades in the field of hepatic organogenesis and regeneration provides milestones in the development of liver regen‐ erative medicine. Moreover, compared to current operative therapies, it is less invasive, is less expensive, and avoids the problem of shortage of donors, immune rejection, and other similar complications. Ideally, liver regenerative medicine seems an ultimate solution for liver cirrhosis.

Liver regenerative medicine uses two key approaches based on cell therapy and tissue/organ engineering. Cell‐based therapy is defined as the transplantation of cells from different sources with or without differentiation to improve liver function [6]. Transplantation of mature hepa‐ tocytes and liver stem/progenitor cells (LSPCs) from allogeneic sources is already in clinical trials. However, current research is intended to overcome the problem of immune rejection associated with allogeneic sources and focuses on therapies based on generation of autolo‐ gous hepatocytes from MSCs and induced pluripotent stem cells (iPSCs) [5]. Elucidation of cell type, which can be successfully differentiated into functional and transplantable hepato‐ cytes or liver progenitor cells, is another major task under study [7]. Furthermore, researchers are trying to refine protocols for proliferation, differentiation, and storage of these cells to have them in plenty and always ready to be transplanted.

Second strategy mainly covers the area of liver tissue/organ engineering, engraftment, and monitoring in patients. Ongoing therapeutic approaches in tissue engineering include implant‐ able constructs of hepatic tissues and whole organ. For the construction of hepatic tissues, nat‐ ural and synthetic bioactive scaffolds are designed [5]. Nanotechnology and microchip devices are contributing a lot in this lane. Moreover, whole organ engineering is also in great focus to escape end‐stage liver diseases. However, determination of ideal cell types, cell volume, and optimal seeding techniques is yet to be discovered [8, 9].

This chapter deals with the scope of liver regenerative medicine in the treatment of liver cir‐ rhosis. Different operative and proposed therapies along with their pros and cons are the major focus of this section and are reviewed in detail.

## **2. Hepatic organogenesis**

leading to liver cirrhosis. Liver cirrhosis is irreversible and mostly ends up with complete loss of liver function/end‐stage liver failure. End‐stage liver failure with high rates of morbid‐ ity and mortality poses a significant threat to human health as well as economy throughout the world [1]. As current pharmacological treatments are inefficient to reverse this loss, liver transplantation is the only effective lifesaving option. Since the first liver transplantation in 1963, the number of cases requiring transplantation are considerably increasing with the pas‐ sage of time. Despite the success of liver transplantation, there is a gap between demand and supply. Only 30–50% of annual liver donation desires are fulfilled and at least about 15% patients die while being on the waiting list [2, 3]. Besides scarcity of liver donors, high cost, postoperative graft rejection, and long‐term immune‐suppression are few more serious constraints associated with liver transplant [4]. Therefore, it is crucial to look for effective and

Advancements in the field of regenerative medicine open up new horizons and arising hope in the treatment of irreversibly damaged liver cirrhosis. Liver regenerative medicine mainly emphasizes on the establishment of new therapies to either functionally restore the chronically damaged liver tissue or to develop the entire new organ [5]. Elucidation of cel‐ lular and molecular mechanisms during the last couple of decades in the field of hepatic organogenesis and regeneration provides milestones in the development of liver regen‐ erative medicine. Moreover, compared to current operative therapies, it is less invasive, is less expensive, and avoids the problem of shortage of donors, immune rejection, and other similar complications. Ideally, liver regenerative medicine seems an ultimate solution for

Liver regenerative medicine uses two key approaches based on cell therapy and tissue/organ engineering. Cell‐based therapy is defined as the transplantation of cells from different sources with or without differentiation to improve liver function [6]. Transplantation of mature hepa‐ tocytes and liver stem/progenitor cells (LSPCs) from allogeneic sources is already in clinical trials. However, current research is intended to overcome the problem of immune rejection associated with allogeneic sources and focuses on therapies based on generation of autolo‐ gous hepatocytes from MSCs and induced pluripotent stem cells (iPSCs) [5]. Elucidation of cell type, which can be successfully differentiated into functional and transplantable hepato‐ cytes or liver progenitor cells, is another major task under study [7]. Furthermore, researchers are trying to refine protocols for proliferation, differentiation, and storage of these cells to

Second strategy mainly covers the area of liver tissue/organ engineering, engraftment, and monitoring in patients. Ongoing therapeutic approaches in tissue engineering include implant‐ able constructs of hepatic tissues and whole organ. For the construction of hepatic tissues, nat‐ ural and synthetic bioactive scaffolds are designed [5]. Nanotechnology and microchip devices are contributing a lot in this lane. Moreover, whole organ engineering is also in great focus to escape end‐stage liver diseases. However, determination of ideal cell types, cell volume, and

This chapter deals with the scope of liver regenerative medicine in the treatment of liver cir‐ rhosis. Different operative and proposed therapies along with their pros and cons are the

operative alternate approaches of liver transplantation.

234 Liver Cirrhosis - Update and Current Challenges

have them in plenty and always ready to be transplanted.

optimal seeding techniques is yet to be discovered [8, 9].

major focus of this section and are reviewed in detail.

liver cirrhosis.

Zygote is the only totipotent structure that leads to the development of blastocyst. Blastocyst carries both embryonic and extraembryonic (inner cell mass) cell population. Inner cell mass (ICM) forms three germ layers: exoderm, mesoderm, and endoderm. Embryonic liver develops from the endodermal layer during ventral foregut closure in the midgut [10]. Cells residing in the hepatic bud are bipotent and are called hepatoblasts. Hepatoblasts are columnar in shape, release α‐fetoprotein, and differentiate into mature hepatocytes and cholangiocytes [11].

Wingless type (wnt) signaling pathway, together with activin‐A, plays a crucial role in the establishment of endoderm during primitive streak formation and differentiation of liver precursor cells toward hepatoblasts [12, 13]. Other key factors involved in hepatic fate deter‐ mination are fibroblast growth factors (FGFs) released from cardiac mesoderm and bone mor‐ phogenetic proteins (BMPs) released by septum transversum mesenchyme [3]. Furthermore, oncostatin M and hepatocyte growth factor (HGF) control the differentiation of hepatoblasts toward hepatocytes [14], whereas Jagged‐Notch signaling pathway is responsible for the development of cholangiocytes [15].

Gradually, as the liver development proceeds toward the final stages of maturation, hep‐ atoblast number reduces markedly. Liver becomes populated with mature and unipotent hepatocytes and cholangiocytes. The remainder resident cells of liver, that is, Kupffer cells, stellate cells, and endothelium, are mesodermal in origin. Majority of the liver functions are performed by hepatocytes. On the onset of any hepatic insult, adult liver cells undergo apoptosis that calls for the replacement of lost cells or in other words liver regeneration. The schematic diagram of liver organogenesis from endodermal layer along with important molecular signaling pathways involved in activation or suppression of each step has been represented in **Figure 1**.

**Figure 1.** Schematic diagram of liver organogenesis. Molecular signals involved in the activation of each stage are indicated in the boxes occuring at various steps of liver organogenesis.

### **3. Liver regeneration**

Elucidation of the cellular and molecular mechanisms involved in liver regeneration provides vital scientific grounds for liver regenerative medicine. Depending upon the origin of liver damage, different kinds of repair mechanisms are operative [16]. Various surgical and toxin‐ mediated injury models for liver regeneration have been established so far. One of the estab‐ lished and utterly studied model of regeneration is rodent partial hepatectomy [17]. In partial hepatectomy model, liver can regenerate to its normal size in 3–10 days even if two‐thirds of its mass is surgically removed. A fine coordination of cellular and molecular events occurs in the regeneration process of partial hepatectomy. Robust hepatocyte replication followed by hypertrophy has been revealed as an underlying cellular mechanism in partial hepatec‐ tomy recovery. This vigorous change in hepatocytes is also accompanied by alteration of gene expression patterns, instigation of transcription factors, and release of growth signals. More than 100 genes are activated in an early response manner. At least 40% of these early response genes are activated by interleukin‐6 (IL‐6) signaling which itself is activated by tumor necrosis factor‐α (TNF‐α)‐mediated NFκB (nuclear factor kappa‐B) activation [18, 19]. The recovery of liver mass and function of living donor and recipient of liver transplantation in humans seems to adopt a similar track.

Besides utilizing mature hepatocytes for liver regeneration, another likely approach is the use of liver progenitor cells (LSPCs). They are capable of converting into different cell lines found in liver, that is, hepatocytes, oval cells, and stellate cells [20]. LSPCs got experimen‐ tal and clinical support when they were overproliferated in case of induced liver injury by acetaminophen and slowly proliferated in case of liver cirrhosis [21, 22]. At present, the main focus is on the regenerative capacity of LSPCs when hepatocytes run out of their regenerative potential. LSPCs are also proved potential progenitor cells of biliary epithelium in vitro, but no specific LSPC markers are identified as yet. It seems that LSPCs are driven by the activation of certain genes and the combination of growth factors. Crucially important genes include Leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (LGR5) and the cytokine tumor necrosis factor‐like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis fac‐ tor (TNF) superfamily [23]. Some other mitogenic factors also play a crucial role, for example, HGF, epidermal growth factor (EGF), TGF‐α, and fibroblast growth factors 1 and 2 (FGF1 and FGF2) [24]. However, there is lack of evidence pertaining to in vivo differentiation of LSPCs into hepatocytes. The articles published in 2014 used different methodologies to trace the fate of liver progenitor cells. They utterly rejected the concept of regenerative capability of LSPCs into hepatocytes. Besides, despite lack of proof of the in vivo hepatogenic differentiation of LSPCs, they surely can give rise to hepatocyte‐like cells in vitro [20]. Research in this arena is ongoing and there is a probability that even in mice a part for oval cells/LSPCs in regeneration will be found.

Third major concept in liver regeneration is through extrahepatic cells that is hematopoi‐ etic stem cells (HSCs) and mesenchymal stem cells (MSCs) derived from bone marrow. HSC and MSC from bone marrow reach the liver via blood circulation. These HSCs and MSCs can populate the liver after hepatogenic differentiation [25]. It is proposed that these bone marrow‐derived stem cells are not directly converted into hepatocytes rather they first mix with resident liver cells and then participate in liver repopulation [26]. It has also been sug‐ gested that MSCs with multilineage differentiation potential provide a great variety of cells for nonhematopoietic tissues like liver tissues [27]. Though they are highly heterogeneous in nature, only a little fraction of it contributes to liver regeneration [28]. It is notable that bone marrow cells take part in the regeneration of liver endothelium. Twenty percent of the liver endothelial cells are made by the bone marrow‐derived endothelial cells [29]. There is a need of concerning involvement of bone marrow‐derived stem cells in liver parenchyma regenera‐ tion, for designing the methods for cellular therapy of liver disease [16].

## **4. Cell‐based therapies for regeneration of liver cirrhosis**

Cell‐based therapies are the oldest and most efficient method to regenerate damaged liver. Effective engraftment and proliferation of donor cells in the recipient liver are the main issues of concern for liver regeneration through cell‐based therapy. Depending on the donor source, cells can be of autologous [30], allogeneic, or syngeneic nature [31]. The cells are injected into the recipient through portal vein, peripheral vein [30], and intraspleenic [32] or intraperi‐ toneal route. To enhance the transplantation efficiency, conditioning of recipient liver with partial hepatectomy [33, 34], liver irradiation [35, 36], or portal embolization [37] has been recently proposed. Broadly, cells are categorized into two main categories; stem cells and mature hepatocytes are the potential cell‐based therapies adapted to date in the cure and regeneration of liver cirrhosis [5]. The roles of these cell‐based therapies are shown in **Figure 2** and are discussed one by one in detail in the following section.

#### **4.1. Hepatocytes and liver regeneration**

**3. Liver regeneration**

236 Liver Cirrhosis - Update and Current Challenges

to adopt a similar track.

will be found.

Elucidation of the cellular and molecular mechanisms involved in liver regeneration provides vital scientific grounds for liver regenerative medicine. Depending upon the origin of liver damage, different kinds of repair mechanisms are operative [16]. Various surgical and toxin‐ mediated injury models for liver regeneration have been established so far. One of the estab‐ lished and utterly studied model of regeneration is rodent partial hepatectomy [17]. In partial hepatectomy model, liver can regenerate to its normal size in 3–10 days even if two‐thirds of its mass is surgically removed. A fine coordination of cellular and molecular events occurs in the regeneration process of partial hepatectomy. Robust hepatocyte replication followed by hypertrophy has been revealed as an underlying cellular mechanism in partial hepatec‐ tomy recovery. This vigorous change in hepatocytes is also accompanied by alteration of gene expression patterns, instigation of transcription factors, and release of growth signals. More than 100 genes are activated in an early response manner. At least 40% of these early response genes are activated by interleukin‐6 (IL‐6) signaling which itself is activated by tumor necrosis factor‐α (TNF‐α)‐mediated NFκB (nuclear factor kappa‐B) activation [18, 19]. The recovery of liver mass and function of living donor and recipient of liver transplantation in humans seems

Besides utilizing mature hepatocytes for liver regeneration, another likely approach is the use of liver progenitor cells (LSPCs). They are capable of converting into different cell lines found in liver, that is, hepatocytes, oval cells, and stellate cells [20]. LSPCs got experimen‐ tal and clinical support when they were overproliferated in case of induced liver injury by acetaminophen and slowly proliferated in case of liver cirrhosis [21, 22]. At present, the main focus is on the regenerative capacity of LSPCs when hepatocytes run out of their regenerative potential. LSPCs are also proved potential progenitor cells of biliary epithelium in vitro, but no specific LSPC markers are identified as yet. It seems that LSPCs are driven by the activation of certain genes and the combination of growth factors. Crucially important genes include Leucine‐rich repeat‐containing G‐protein‐coupled receptor 5 (LGR5) and the cytokine tumor necrosis factor‐like weak inducer of apoptosis (TWEAK), a member of the tumor necrosis fac‐ tor (TNF) superfamily [23]. Some other mitogenic factors also play a crucial role, for example, HGF, epidermal growth factor (EGF), TGF‐α, and fibroblast growth factors 1 and 2 (FGF1 and FGF2) [24]. However, there is lack of evidence pertaining to in vivo differentiation of LSPCs into hepatocytes. The articles published in 2014 used different methodologies to trace the fate of liver progenitor cells. They utterly rejected the concept of regenerative capability of LSPCs into hepatocytes. Besides, despite lack of proof of the in vivo hepatogenic differentiation of LSPCs, they surely can give rise to hepatocyte‐like cells in vitro [20]. Research in this arena is ongoing and there is a probability that even in mice a part for oval cells/LSPCs in regeneration

Third major concept in liver regeneration is through extrahepatic cells that is hematopoi‐ etic stem cells (HSCs) and mesenchymal stem cells (MSCs) derived from bone marrow. HSC and MSC from bone marrow reach the liver via blood circulation. These HSCs and MSCs can populate the liver after hepatogenic differentiation [25]. It is proposed that these bone marrow‐derived stem cells are not directly converted into hepatocytes rather they first mix Liver is chiefly composed of hepatocytes. Hepatocyte proliferation plays a distinctive role in liver regeneration under both acute and chronic injury conditions. The unique characteristic

**Figure 2.** Different types of cells and their mode of application for cell‐based therapies of liver cirrhosis. Different types of cells isolated from humans and being used in liver regeneration are shown on the left side of the figure. Each of the cell type has been injected and has recovered liver functions either through only in vitro proliferation (hepatocytes), via differentiation toward hepatocytes (ESCs and iPSCs) or through both (MSCs, LSPCs).

of hepatocytes to proliferate under stress conditions makes them ideal cell type for cell‐based therapies. Primary hepatocytes were the very first type of cells to be used for cell‐based ther‐ apy of liver. Isolated hepatocytes are infused either directly into the liver or into the spleen from where they can migrate to and settle down in the liver. The hepatocyte transplantation has shown to considerably improve the hepatic functions even in end‐stage liver failure [38]. Typically, hepatocytes are harvested from the livers that are not suitable for transplantation [39]. However, due to problem of immune rejection, it was also tried to isolate hepatocyte from patient's biopsies [40].

Although primary hepatocytes are ideal for use in liver regeneration, this approach is prone to certain limiting factors. Inadequate supply of the required cells, slow in vitro prolifera‐ tion rate [18], dedifferentiation within 72 hours of culturing [41], susceptibility to freeze‐thaw damage, and loss of certain characteristic features in culture conditions are major obstacles that hinder the utilization of these cells for liver regeneration [38]. The isolated primary hepatocytes are of low quantitative value, and an autologous isolation of this cell population involves patients' inconvenience. Typically, hepatocytes are harvested from the livers that are not suitable for transplantation, so the quantitative and qualitative values of obtained cells vary considerably. All of these constraints have played a pivotal role in shifting focus toward alternate cell‐based therapies.

#### **4.2. Stem cells in liver regeneration**

With the therapeutic focus being set on the establishment of personalized medicine and the replacement or regeneration of damaged tissue, stem cell‐based therapies may provide a strong platform. The properties of indefinite cell division and differentiation potential into other cell types make the stem cells an ideal choice for cure and regeneration of liver cirrhosis. Another important property of stem cells is their ability to create and provide a favorable environment for growth of primary hepatocytes and/or hepatocyte‐like cells [5]. Coculturing MSCs with primary hepatocytes results in their improved viability and function by provid‐ ing structural and paracrine trophic support [41–43]. Moreover, stem cell therapy holds great potential especially in the cure of inherited liver diseases, where, together with gene therapy, it may correct metabolic disorders permanently without even using immunosuppressive drugs [5]. Chiefly, two approaches of stem cell‐based liver regeneration are in practice either their direct injection or in vitro differentiation toward hepatocyte‐like cells and transplantation.

Some types of stem cells show efficient growth in vitro, could be a rich pool to supply hepa‐ tocytes/precursor cells, and thus be used largely for transplantation. If the wide availability of human hepatocytes is made possible, this could be a major breakthrough in the treatment of various liver diseases. However, the research work debating good capacity stem cell therapy lack in reproducibility evidence or some of these even have been overoptimistically inter‐ preted. Another important milestone is to decide on the preference of stem and precursor cell types. It is a difficult task to compare different cell types with respect to their reported capac‐ ity of differentiation toward hepatocytes [44]. We therefore discuss the possibilities these cell therapies offer one by one, along with the limitations which are making these feats harder to achieve.

#### *4.2.1. Embryonic stem cells and hepatocyte generation*

of hepatocytes to proliferate under stress conditions makes them ideal cell type for cell‐based therapies. Primary hepatocytes were the very first type of cells to be used for cell‐based ther‐ apy of liver. Isolated hepatocytes are infused either directly into the liver or into the spleen from where they can migrate to and settle down in the liver. The hepatocyte transplantation has shown to considerably improve the hepatic functions even in end‐stage liver failure [38]. Typically, hepatocytes are harvested from the livers that are not suitable for transplantation [39]. However, due to problem of immune rejection, it was also tried to isolate hepatocyte

Although primary hepatocytes are ideal for use in liver regeneration, this approach is prone to certain limiting factors. Inadequate supply of the required cells, slow in vitro prolifera‐ tion rate [18], dedifferentiation within 72 hours of culturing [41], susceptibility to freeze‐thaw damage, and loss of certain characteristic features in culture conditions are major obstacles that hinder the utilization of these cells for liver regeneration [38]. The isolated primary hepatocytes are of low quantitative value, and an autologous isolation of this cell population involves patients' inconvenience. Typically, hepatocytes are harvested from the livers that are not suitable for transplantation, so the quantitative and qualitative values of obtained cells vary considerably. All of these constraints have played a pivotal role in shifting focus toward

With the therapeutic focus being set on the establishment of personalized medicine and the replacement or regeneration of damaged tissue, stem cell‐based therapies may provide a strong platform. The properties of indefinite cell division and differentiation potential into other cell types make the stem cells an ideal choice for cure and regeneration of liver cirrhosis. Another important property of stem cells is their ability to create and provide a favorable environment for growth of primary hepatocytes and/or hepatocyte‐like cells [5]. Coculturing MSCs with primary hepatocytes results in their improved viability and function by provid‐ ing structural and paracrine trophic support [41–43]. Moreover, stem cell therapy holds great potential especially in the cure of inherited liver diseases, where, together with gene therapy, it may correct metabolic disorders permanently without even using immunosuppressive drugs [5]. Chiefly, two approaches of stem cell‐based liver regeneration are in practice either their direct injection or in vitro differentiation toward hepatocyte‐like cells and transplantation.

Some types of stem cells show efficient growth in vitro, could be a rich pool to supply hepa‐ tocytes/precursor cells, and thus be used largely for transplantation. If the wide availability of human hepatocytes is made possible, this could be a major breakthrough in the treatment of various liver diseases. However, the research work debating good capacity stem cell therapy lack in reproducibility evidence or some of these even have been overoptimistically inter‐ preted. Another important milestone is to decide on the preference of stem and precursor cell types. It is a difficult task to compare different cell types with respect to their reported capac‐ ity of differentiation toward hepatocytes [44]. We therefore discuss the possibilities these cell therapies offer one by one, along with the limitations which are making these feats harder to

from patient's biopsies [40].

238 Liver Cirrhosis - Update and Current Challenges

alternate cell‐based therapies.

achieve.

**4.2. Stem cells in liver regeneration**

Differentiation of cultured embryonic stem cells toward hepatocyte‐like cells in vitro appears to be the most studied model of mature hepatocyte generation. In mouse models of liver injury, hepatocyte‐like cells not only recover the liver by proliferation but also provide trophic factors that assist the endogenous hepatic regenerative capability [45]. Human ESCs efficiently form embryoid bodies in suspension cultures forming three germ layers [46]. Hepatocyte iso‐ lation from this heterogeneous cell population is very difficult, suggesting endoderm enrich‐ ment to be a practical option with maximum hepatocyte yield.

A directional differentiation strategy for the generation of functional hepatocytes from embryonic stem cells involves sequential supplementation of various molecular factors (growth factors and cytokines necessary for development of human embryonic liver)‐ enriched growth medium. The molecular factors involved in early embryonic differentiation such as fibroblast growth factor (FGF2/4), bone morphogenetic protein (BMP2/4), activin A and Wnt3 can be used for endoderm enrichment from cultivated embryoid bodies [44, 46]. FGF2/4 stimulates the development of hepatoblasts from cultured ESCs and the generation of mature hepatocytes, whereas HGF plays a supportive role in hepatocyte generation from hepatoblasts. Dexamethasone (glucocorticoid hormone) induces the production of adult hepatocyte‐specific proteins. This strategy ensures an 80–90% hepatocyte yield. Recently, Wang et al. established a polymer‐modified nanoparticle‐based sustained delivery system for growth factors to direct stem cell differentiation into hepatocytes [47]. Their approach can help to overcome the limitations linked with current models and make sure efficient delivery of growth factors to improve ESC differentiation toward a hepatocyte‐like lineage.

The final and most important step in this strategy involves isolation of absolute hepatocyte population from a heterogeneous mixture containing other hepatic precursors and immature hepatocytes. Basma et al. used asialoglycoprotein receptor ASGPR1 (hepatocyte‐specific cell surface marker) expression based sorting to enrich the pure hepatocyte populations [48]. To enhance the isolation efficiency of hepatocytes based on ASGPR1, fluorescent‐labeled or mag‐ net‐coated antibodies are further proposed [49]. However, further research is required to be performed to isolate definitive hepatocyte population or to obtain a relatively absolute ratio of hepatocytes from ESCs [50].

Despite their success stories, there are a number of ethical issues concerning the use of human ESCs in liver regenerative medicine [50]. Furthermore, pluripotency of these cells is very dif‐ ficult to handle leading to an uncontrolled regenerative potential. Above all, putative tumori‐ genicity associated with transplantation of ESCs proves to be an additional barrier for their clinical application [49–50].

#### *4.2.2. Bone marrow stem cells (BMSCs)*

In bone marrow, three different pluripotent cell populations, that is hematopoietic stem cells (HSCs), MSCs, and multipotent adult progenitor cells (MAPCs)/endothelial progenitor cells (EPCs), are present [51]. Peripheral blood, umbilical cord blood, and synovial fluid are addi‐ tional sources of HSCs and MSCs. HSCs and MSCs can be advantageous cell sources for liver regeneration as compared to hepatocytes since they can be obtained relatively easily from blood and bone marrow of live donors. Since BMSCs are immune‐modulators, a reduced chance of graft rejection is an additional property of these stem cells [47, 51]. In clinical trials, patients with autologous BMSC (CD34+ cell) transplantation had no procedure‐related com‐ plications and showed improved quality of life [30]. MSCs have proven reliable for treatment of liver cirrhosis in phase I and phase II clinical trials as shown in **Table 1**.



regeneration as compared to hepatocytes since they can be obtained relatively easily from blood and bone marrow of live donors. Since BMSCs are immune‐modulators, a reduced chance of graft rejection is an additional property of these stem cells [47, 51]. In clinical trials,

plications and showed improved quality of life [30]. MSCs have proven reliable for treatment

**Administration** 

**route**

Liver cirrhosis 9 intraportal 10 months

of liver cirrhosis in phase I and phase II clinical trials as shown in **Table 1**.

**patients**

cell) transplantation had no procedure‐related com‐

**Outcomes/clinical significance**

and clinical improvement

function and MELD score

safe procedure; improved liver function

AST, ALT, PT; improved ALB, PC, PT, INR

side effects; histological improvement; improved CP score

effects; improved ALB, CP scores

score, BIL, ALB, and PC

effects; decreased serum ALP and

GGT

side effects; improved liver function and MELD score; reduced ascites

Longer survival [40]

**References**

[74]

[32]

[75]

[76]

[77]

[78]

[79]

[80]

[81]

**Follow‐up period**

in only one patient

2 hepatic artery 12 months Biochemical

25 hepatic artery 6 months Improved liver

4 peripheral vein 12 months Well‐tolerated and

20 intrasplenic 6 months Decreased TBIL,

11 hepatic artery 12 months No significant

intravenous 6 months Improved MELD

intravenous 12 months No significant

7 peripheral vein 12 months No obvious side

Liver cirrhosis 9 peripheral vein 6 months No major adverse

patients with autologous BMSC (CD34+

240 Liver Cirrhosis - Update and Current Challenges

**Cell source Liver cirrhosis No. of** 

Advanced cirrhosis

End‐stage liver cirrhosis

liver cirrhosis

post‐HCV liver cirrhosis

post‐HCV liver cirrhosis

cirrhosis

Post‐HBV decompensated liver cirrhosis

UC‐MSCs Primary biliary

10: control 15: treated

15: control 30: treated

Alcoholic cirrhosis

Hepatocytes (autologous)

EpCAM+

liver‐SCs

Autologous BM‐MSCs

BM‐MSCs (Differentiated *vs* undifferentiated)

Fetal

BM‐MSCs Decompensated

EpCAM: Epithelial cell adhesion molecule; GGT: γ‐glutamyl transferase; ALT: Alanine aminotransferase; TBIL: Total bilirubin; AST: Aspartate aminotransferase; CP: Child‐Pugh; HGF: Hepatocyte growth factor; HCV: Hepatitis C virus; PT: Prothrombin time; ALB; Albumin; PC: Platelet count; INR: International normalized ratio; MELD: Model for end‐ stage liver diseases; ALP: Alkaline phosphatase; UC‐MSC: Umbilical cord blood‐mesenchymal stem cells; G‐CSF: Granulocyte‐colony‐stimulating factor; BM‐MSCs: Bone marrow‐mesenchymal stem cells.

**Table 1.** Clinical trials of cell‐based therapies along with their route of administration, follow‐up, and outcomes.

Hematopoietic stem cells originating from bone marrow are efficient stem cell population that migrates to the site of injury and participate in the repopulation of damaged tissue. In liver regeneration, this stem cell population is postulated to contribute based on the cell fusion capa‐ bility of the BMSCs [52, 53] rather than cellular differentiation. In murine hepatectomy models, BMSCs were found to fuse with hepatocytes, and the resultant hybrid cells were shown to be responsible for triggering proficient liver regenerative reaction [54]. Therapeutic mechanisms of MSCs are reported to be more clear as compared to those of HSCs. MSCs not only reduce inflammation and fibrosis but they also increase liver regenerative response in a much rapid manner than HSCs [55]. CD34 is reported to be an efficient cellular marker for the isolation of HSCs [30]. However, these cells have showed profibrogenic potential in some cases [56].

Despite wide use in preclinical setting and clinical trials, the BMSCs have to be evaluated extensively for their potential role in liver regeneration before being applied to the wide clin‐ ical utilization. Tumorigenicity of MSCs is another constraint that needs to be considered while using this stem cell population in clinical application [57].

#### *4.2.3. Adipose‐derived stem cells (ADSCs)*

Adipose tissue is another source of MSCs used for hepatic regeneration. ADSCs seem to be pluripotent and have the potential to differentiate into cells of multiple germ lines such as bone, nerve, heart, and adipose tissue. These cells are advantageous over BMSCs because of their higher in vitro proliferation activity and differentiation potential [58]. The sufficient availability of adipose tissue from most patients with no substantial defects renders ADSCs an efficient alternative source of stem cells for liver regeneration [59]. Differentiation of ADSCs into functional hepatocytes involves activation of Wnt/beta‐catenin signaling through glyco‐ gen synthase kinase 3 inhibitors [60]. Further research is needed to evaluate the potential of this stem cell lineage in liver regenerative setups.

#### *4.2.4. Liver stem/progenitor cells (LSPCs)*

Hepatoblasts being bipotent are capable of self‐renewal and differentiation into cholangio‐ cytes and hepatocytes. In contrast to ESCs and MSCs, both of which need to go through sequential differentiation to develop into mature hepatocytes, LSPCs have a destined fate. Hence, they carry significant potential to be used in liver regenerative medicine. LSPCs can undergo several rounds of proliferation. These cells have the potential to differentiate into hepatic and biliary cell lineages and to repair the damaged liver tissue [50, 61]. LSPCs are thought to be the cells that do not contribute to the routine liver yields. Instead, they appear in advance stages of liver injury such as primary biliary cirrhosis and nonalcoholic cirrhosis [21]. Many properties of embryonic hepatoblasts are shared by LSPCs. Certain surface mark‐ ers help in selective isolation of LSPCs via immune selection. They express epithelial cell adhesion molecules (EpCAM) and have been isolated against this surface marker [11] from fetal as well as adult human liver [62]. Differentiation of EpCAM‐positive cells can yield both hepatocytes and cholangiocytes [63, 64]. Clinical trials of EpCAM‐positive LSPCs are given in **Table 1**.

LSPCs, on the other hand, have certain limitations which hinder their application in liver regen‐ erative medicine. First of all, these cells are present in a very small quantity in the adult human liver making it unproductive to isolate them on the basis of their markers. Our research group had addressed this problem in a recently published study, where BMSCs were differentiated toward oval cell‐like cells. These oval cell‐like cells were comparable to control oval cells in their efficiency to reduce liver injury [65]. Another major issue associated with LSPCs is their great potential to induce hepatic tumorigenicity. Presently, this is a major limiting factor for their utilization in liver therapeutics and regenerative medicine. Notably, human liver progeni‐ tor cells have been found to be present and contributing in the development of nonalcoholic steatohepatitis in pediatric and adult human patients. They are supposed to be playing fibro‐ genic role in such cases as reported by Sobaniec‐Łotowska et al. [66]. Comprehensive research at preclinical level is required to probe into these issues properly to understand the appropri‐ ateness of these cells for clinical trials.

### *4.2.5. Induced pluripotent stem cells (iPSCs)*

inflammation and fibrosis but they also increase liver regenerative response in a much rapid manner than HSCs [55]. CD34 is reported to be an efficient cellular marker for the isolation of HSCs [30]. However, these cells have showed profibrogenic potential in some cases [56].

Despite wide use in preclinical setting and clinical trials, the BMSCs have to be evaluated extensively for their potential role in liver regeneration before being applied to the wide clin‐ ical utilization. Tumorigenicity of MSCs is another constraint that needs to be considered

Adipose tissue is another source of MSCs used for hepatic regeneration. ADSCs seem to be pluripotent and have the potential to differentiate into cells of multiple germ lines such as bone, nerve, heart, and adipose tissue. These cells are advantageous over BMSCs because of their higher in vitro proliferation activity and differentiation potential [58]. The sufficient availability of adipose tissue from most patients with no substantial defects renders ADSCs an efficient alternative source of stem cells for liver regeneration [59]. Differentiation of ADSCs into functional hepatocytes involves activation of Wnt/beta‐catenin signaling through glyco‐ gen synthase kinase 3 inhibitors [60]. Further research is needed to evaluate the potential of

Hepatoblasts being bipotent are capable of self‐renewal and differentiation into cholangio‐ cytes and hepatocytes. In contrast to ESCs and MSCs, both of which need to go through sequential differentiation to develop into mature hepatocytes, LSPCs have a destined fate. Hence, they carry significant potential to be used in liver regenerative medicine. LSPCs can undergo several rounds of proliferation. These cells have the potential to differentiate into hepatic and biliary cell lineages and to repair the damaged liver tissue [50, 61]. LSPCs are thought to be the cells that do not contribute to the routine liver yields. Instead, they appear in advance stages of liver injury such as primary biliary cirrhosis and nonalcoholic cirrhosis [21]. Many properties of embryonic hepatoblasts are shared by LSPCs. Certain surface mark‐ ers help in selective isolation of LSPCs via immune selection. They express epithelial cell adhesion molecules (EpCAM) and have been isolated against this surface marker [11] from fetal as well as adult human liver [62]. Differentiation of EpCAM‐positive cells can yield both hepatocytes and cholangiocytes [63, 64]. Clinical trials of EpCAM‐positive LSPCs are given

LSPCs, on the other hand, have certain limitations which hinder their application in liver regen‐ erative medicine. First of all, these cells are present in a very small quantity in the adult human liver making it unproductive to isolate them on the basis of their markers. Our research group had addressed this problem in a recently published study, where BMSCs were differentiated toward oval cell‐like cells. These oval cell‐like cells were comparable to control oval cells in their efficiency to reduce liver injury [65]. Another major issue associated with LSPCs is their great potential to induce hepatic tumorigenicity. Presently, this is a major limiting factor for

while using this stem cell population in clinical application [57].

*4.2.3. Adipose‐derived stem cells (ADSCs)*

242 Liver Cirrhosis - Update and Current Challenges

this stem cell lineage in liver regenerative setups.

*4.2.4. Liver stem/progenitor cells (LSPCs)*

in **Table 1**.

The establishment of iPSCs by reprogramming somatic cells through certain transcription factors (Oct‐3/4, Sox2, Nanog, c‐Myc, Klf‐4) has proven a potential new source of stem cells. These cells exhibit properties essential for ESCs and have the potential to differentiate into the derivatives of all three germ layers [67]. However, iPSCs avoid the ethical issues related to ESCs since no human embryo is used for their production [3]. iPSCs being autologous in nature also evade the problem of immune rejection. Although there are unlimited sources for iPSCs generation, to ensure a relatively homogeneous hepatocyte culture, the use of hepato‐ cytes or/and other endodermal cells is recommended. It can play an important role as cells carry an "epigenetic memory" allowing the iPSCs to differentiate toward cells of definitive germ layer [68].

Permanent retroviral integration, a process which was initially used by Takashi and cowork‐ ers in 2007 [69] is one of the earliest methods used for iPSCs production. With advancement in the field, it is possible to generate iPSCs without using retroviral transfection. Nowadays, a number of methods such as excisable viral transfection [70], microRNA transfection [71], episomal plasmid transfection [72], and mRNA transfection [73] are being harnessed for the production of functionally efficient iPSCs. Once generated, iPSCs can be directed to differen‐ tiate toward definitive endoderm which will differentiate into hepatoblasts and finally into hepatocytes in a sequential manner involving various growth factors, cytokines, and signal‐ ing pathways as described previously in this chapter. The resultant hepatocyte‐like cells are more like fetal hepatocytes rather than mature hepatocytes, a phenomenon shared by all the stem cell‐generated hepatocytes [3]. Although an efficient source of autologous transplanta‐ tion, iPSCs‐derived hepatocytes have certain shortcomings as well.

## **5. Tissue engineering and liver cirrhosis**

Cell‐based therapies have shown promising results in the improvement of liver cirrhosis. However, inefficient engraftment of cells due to surrounding conditions of diseased liver results in variable outcomes [3]. Tissue engineering, a recent advancement in liver regen‐ erative medicine, is dedicated in deriving the ways to escape the problems associated with direct cell‐based therapies. It mainly focuses on the development of biocompatible scaffolds and extracorporeal liver devices suitable for either in vitro or in vivo applications. Schematic representation of key approaches used for liver tissue engineering is shown in **Figure 3** and discussed in detail with their merits and relevant complications in the following section.

**Figure 3.** Schematic diagram of liver tissue engineering. Solid lines show the approaches already ongoing whereas dotted lines indicate the proposed mechanisms.

#### **5.1. Generation of bioactive scaffolds**

Bioactive scaffolds are those that have the ability to elicit cell growth and differentiation. In modern tissue engineering, bioactive scaffolds are so much advantageous as they mimic the natural ECM environment of the liver. One of the major components of these scaffolds is a structural protein collagen normally found in skin, bone, and cartilage [90]. Collagen highly supports attachment, proliferation, differentiation, growth, and migration of cells. Further, collagen‐based bioscaffolds have shown in vitro differentiation of embryoid bod‐ ies derived from embryonic stem cell into hepatocyte‐like cells [91, 92]. Hyaluronic acid is another important component of the extracellular matrix. It is involved in the regulation of cell proliferation and expansion. The immature and mature hepatocytes of fetal and adult liver cells express surface receptors for hyaluronic acid, that is CD44 [93]. By utilizing this property of hepatocytes, hydrogels consisting of hyaluronic acid and its derivatives are syn‐ thesized possessing more adhesive power for hepatocytes. They can retain viability of hepa‐ tocytes for 4 weeks [93].

Other natural biomaterials being utilized in the formation of bioactive scaffolds are alginate, chitin, chitosan, silk, matrigel, and sponge. Its best example is silk‐fibroin‐based microfluidic devices that successfully supported the growth and differentiation of HepG2 cells [94]. Hepatic organoids and smaller parts of tissues can be grown from porcine hepatocytes on the matrix, consisting of albumin and chitosan (a deacetylated form of chitin) [95]. Scaffold containing chi‐ tosan nanofibers associated with the glucose residues showed prolonged metabolic activity of cluster of cells originated from hepatocytes [96]. Hydrogels formed by the natural biomaterials such as alginate and matrigels are more biocompatible and improve the seeding potency of hepatocytes. The basal membranes of murine chondrosarcoma are used for extraction of pro‐ teins (laminin, heparan sulfate proteoglycan, collagen type IV) that are used in the formation of matrigels. Hepatocytes initially started to grow in scaffolds containing matrigels into shapeless clusters of cells followed by their implantation in natural organ [97].

However, it has not yet been recognized that which composition would provide the best physicochemical characteristics for defined growth pattern of hepatocytes. Moreover, due to xenogeneic and tumorigenic origin of matrigels, they are not considered good for tissue engi‐ neering of liver. Although utilization of natural polymers in three‐dimensional (3D) scaffolds creates some histoarchitectural features that help a lot in the generation of cell‐to‐cell and cell‐to‐matrix interactions, uncontrollable physicochemical properties, degradability, lack of regenerative ability, and inconsistent mechanical properties halt its clinical implication.

#### **5.2. Synthetic polymers used in liver tissue engineering**

**5.1. Generation of bioactive scaffolds**

dotted lines indicate the proposed mechanisms.

244 Liver Cirrhosis - Update and Current Challenges

tocytes for 4 weeks [93].

Bioactive scaffolds are those that have the ability to elicit cell growth and differentiation. In modern tissue engineering, bioactive scaffolds are so much advantageous as they mimic the natural ECM environment of the liver. One of the major components of these scaffolds is a structural protein collagen normally found in skin, bone, and cartilage [90]. Collagen highly supports attachment, proliferation, differentiation, growth, and migration of cells. Further, collagen‐based bioscaffolds have shown in vitro differentiation of embryoid bod‐ ies derived from embryonic stem cell into hepatocyte‐like cells [91, 92]. Hyaluronic acid is another important component of the extracellular matrix. It is involved in the regulation of cell proliferation and expansion. The immature and mature hepatocytes of fetal and adult liver cells express surface receptors for hyaluronic acid, that is CD44 [93]. By utilizing this property of hepatocytes, hydrogels consisting of hyaluronic acid and its derivatives are syn‐ thesized possessing more adhesive power for hepatocytes. They can retain viability of hepa‐

**Figure 3.** Schematic diagram of liver tissue engineering. Solid lines show the approaches already ongoing whereas

Other natural biomaterials being utilized in the formation of bioactive scaffolds are alginate, chitin, chitosan, silk, matrigel, and sponge. Its best example is silk‐fibroin‐based microfluidic devices that successfully supported the growth and differentiation of HepG2 cells [94]. Hepatic In comparison to natural biomaterials used in tissue engineering, synthetic materials pro‐ vide a wide range of properties and a better control over them. Their biocompatibility and biodegradability can be tuned easily. Scaffolds containing biodegradable polymers facilitate regeneration, transplantation, and degradation of cells on time. Commonly used biodegrad‐ able polymers are polylactic acid, polyglycolic acid, polyanhydrides, polyfumarates, polyor‐ thoesters, polycaprolactones, poly‐ L–lactic acid, and polycarbonates [98].

A synthetic chemical polyglycolate–polylactate used in 3D scaffolds can turn fetal hepato‐ blasts to mature hepatocytes [99–101]. The main limitations of polyglycolate–polylactate are chemical unpredictability, surface corrosion, and hydrophobicity [102]. However, chemical instability of poly (alpha‐hydroxy) acids results in the formation of hydrolysis products, which can induce inflammatory responses. The chemical modification of polymers (e.g. the incorporation of proteins and special bioactive domains) increases the biocompatibility of bio‐ engineered matrices and improves scaffold adhesion properties stimulating cell attachment and migration, thereby, facilitating liver tissue repair [103]. 3D hepatocyte cultures can also be grown successfully in polyurethanes. Polyurethane foams are used to grow hepatocytes and hepatocyte‐like cells in bioreactors. Highly functional multicellular structures are formed within the pores of these polyurethane foams [104]. Because of these characteristic polyure‐ thane foams are widely used in 3D scaffolds for the production of bioartificial liver [105].

#### **5.3. Implementation of nanotechnology and microchip devices in tissue engineering**

Nanotechnology and microchip devices have tremendous use in liver tissue engineering. Microfluidic devices containing very small volumes of cells, effector molecules, ECM, and so on are used to produce natural biochemical environment around the cells so that they may behave as they do in natural organ [106]. Using the microbioreactors, microcapsule fabrication is done that leads to the encapsulation of hepatic cells and their precursors. In these special kinds of bioreactors, the regular supply of oxygen, water, and nutrients is ensured and metabolic wastes are eliminated. These capsules are made of polydimethylsiloxane and its derivatives because they are highly permeable to water. The polydimethylsiloxane capsules and microspheres of alginate have showed efficient growth of encapsulated hepatocytes that were seeded on them due to its radical perfusion properties. Due to its remarkable properties, polydimethylsiloxane is a promising tool for bioartificial liver system [72].

To estimate cytotoxic effects of drugs on liver cells, 3D microfluidic cell panels have also been introduced. These panels create the natural environment for cells as they are made up of porous hydrogels and are lined with hepatocytes. These pores are taken as capillaries by the cells. Various pharmacokinetic models are being studied with the help of these panels [107, 108].

Speaking collectively, complex microarchitecture of liver tissues having proper cell to cell interactions and supply of cells with oxygen and nutrients are produced from biologically produced microorgans of liver. These microorgans are produced ultimately from bioactive microscaffolds; 3D hepatocyte panels [109].

#### **5.4. Organ‐based regeneration of liver**

The development of whole organ using different techniques in tissue engineering is remark‐ able and this decreases the problems related to shortage of donor organs for transplant and immunosuppression. In order to build a functional liver organ, the first and foremost needed is a scaffold. Among many of the trialed materials for scaffolds, porcine/murine‐based scaf‐ folds have proved better. Second, what is needed is the presence of extracellular matrix in the scaffolds to provide the hepatocytes with their niche for their optimal growth and regulation of cellular behaviors [110, 111].

Complete decellularization of native organ is achieved via detergent perfusion for 24–48 hours, in order to get a xenogeneic scaffold. A point that must be mentioned while decellu‐ larization is: ECM should not be damaged and it should have under 50 ng double‐stranded DNA/mg of ECM to avoid immune rejection [112]. After decellularization, recellularization of xenogenic scaffold with highly functional hepatocytes is done. These cells are obtained either from deceased donor grafts or from partial hepatectomy. However, it is difficult to obtain an appropriate volume of cells. The adult hepatocytes are not considered good for organ regeneration because they show poor in vitro proliferation. Fetal liver cells show high in vitro rate of proliferation but they are not easy to obtain. The human‐derived cell lines that show exponential growth in vitro also cannot be used for implantable organs as they pose the threat of metastasis [113, 114]. Porcine hepatocytes remained successful in BAL system but due to immunogenic rejection they cannot be used for organ bioengineering [5]. Human‐derived autologous stem cells, that is iPSCs, are capable of producing liver‐specific proteins but they produce the albumin at a lower rate than in adult human liver so they are also not a good choice. However, human bone marrow cells are showing promising results in vitro, though they are not yet tested clinically [115].

The recellularization of scaffolds fitted in the tissue cultures of organ chambers is done either by direct parenchymal injections or by single or multistep perfusion in physiological pressure. As a proof of whole liver decellularization and recellularization concept a rat model was utilized for the proliferation of adult rat hepatocytes. Proliferation was confirmed by different markers. Ninety percent of hepatectomized rat models that were given spheroid tissue‐engineered liver showed an increased survival period from 16 to 72 hours. But to their dismay, the rats died of the small‐for‐size syndrome [116, 117].

Besides facing problem in the selection of most suitable cell lines, another hurdle is to develop a vascular network for the support of cell aggregates [118]. Organ bioengineering offers a hopeful way to get out of complications associated with liver cirrhosis. The best scaffold onto which organ is tissue engineered is a decellularized xenogenic scaffold having intact network of ECM. Studies are being focused on the determination of ideal cell types for humans. Deep research is also going on to find the optimal cell seeding techniques and cell volume required to sustain necessary functions [5].

## **6. Conclusion**

that leads to the encapsulation of hepatic cells and their precursors. In these special kinds of bioreactors, the regular supply of oxygen, water, and nutrients is ensured and metabolic wastes are eliminated. These capsules are made of polydimethylsiloxane and its derivatives because they are highly permeable to water. The polydimethylsiloxane capsules and microspheres of alginate have showed efficient growth of encapsulated hepatocytes that were seeded on them due to its radical perfusion properties. Due to its remarkable properties, polydimethylsiloxane

To estimate cytotoxic effects of drugs on liver cells, 3D microfluidic cell panels have also been introduced. These panels create the natural environment for cells as they are made up of porous hydrogels and are lined with hepatocytes. These pores are taken as capillaries by the cells. Various pharmacokinetic models are being studied with the help of these panels [107, 108].

Speaking collectively, complex microarchitecture of liver tissues having proper cell to cell interactions and supply of cells with oxygen and nutrients are produced from biologically produced microorgans of liver. These microorgans are produced ultimately from bioactive

The development of whole organ using different techniques in tissue engineering is remark‐ able and this decreases the problems related to shortage of donor organs for transplant and immunosuppression. In order to build a functional liver organ, the first and foremost needed is a scaffold. Among many of the trialed materials for scaffolds, porcine/murine‐based scaf‐ folds have proved better. Second, what is needed is the presence of extracellular matrix in the scaffolds to provide the hepatocytes with their niche for their optimal growth and regulation

Complete decellularization of native organ is achieved via detergent perfusion for 24–48 hours, in order to get a xenogeneic scaffold. A point that must be mentioned while decellu‐ larization is: ECM should not be damaged and it should have under 50 ng double‐stranded DNA/mg of ECM to avoid immune rejection [112]. After decellularization, recellularization of xenogenic scaffold with highly functional hepatocytes is done. These cells are obtained either from deceased donor grafts or from partial hepatectomy. However, it is difficult to obtain an appropriate volume of cells. The adult hepatocytes are not considered good for organ regeneration because they show poor in vitro proliferation. Fetal liver cells show high in vitro rate of proliferation but they are not easy to obtain. The human‐derived cell lines that show exponential growth in vitro also cannot be used for implantable organs as they pose the threat of metastasis [113, 114]. Porcine hepatocytes remained successful in BAL system but due to immunogenic rejection they cannot be used for organ bioengineering [5]. Human‐derived autologous stem cells, that is iPSCs, are capable of producing liver‐specific proteins but they produce the albumin at a lower rate than in adult human liver so they are also not a good choice. However, human bone marrow cells are showing promising results in vitro, though

The recellularization of scaffolds fitted in the tissue cultures of organ chambers is done either by direct parenchymal injections or by single or multistep perfusion in physiological

is a promising tool for bioartificial liver system [72].

246 Liver Cirrhosis - Update and Current Challenges

microscaffolds; 3D hepatocyte panels [109].

**5.4. Organ‐based regeneration of liver**

of cellular behaviors [110, 111].

they are not yet tested clinically [115].

In conclusion, the field of regenerative medicine has taken a successful initiative toward the ultimate solution of end‐stage liver diseases. Particularly, the dynamism of various cell‐based therapies has arisen much hope and facilitated the development of more challenging tissue engineering. Initially, tissue engineering focused on the use of natural and synthetic scaffolds to grow hepatocytes and develop liver tissues. Currently, much work is ongoing to create liver microorgans to organoids. Crucial aim of future research is to construct whole bioengi‐ neered liver. In this regard, the use of decellularized livers has been proposed to create liver organoids leading to the construction of whole bioengineered liver. However, organ bioen‐ gineering faces the problems of selection of suitable cell type and appropriate development of a vascular network, which will support cell aggregates. Major challenges associated are the determination of suitable cell type, optimal cell volume, and seeding techniques required to endure essential hepatic functions. The current scenario propels to conduct much more experimental work to successfully construct whole bioengineered liver and its effective clini‐ cal applications to replace liver transplantation.

## **Author details**

Asima Tayyeb1 \*, Fareeha Azam<sup>2</sup> , Rabia Nisar1 , Rabia Nawaz<sup>4</sup> , Uzma Qaisar<sup>1</sup> and Gibran Ali<sup>3</sup>


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## **Chapter 13**

## **The Promising Role of Anti-Fibrotic Agent Halofuginone in Liver Fibrosis/Cirrhosis**

## Berna Karakoyun

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Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/intechopen.68641

#### **Abstract**

Liver fibrosis is a complex inflammatory and fibrogenic process that results from chronic liver injury and represents an early step in the progression of cirrhosis. Several cell types [hepatic stellate cells (HSCs), hepatocytes, liver sinusoidal endothelial cells (LSECs), and Kupffer cells (KCs)], cytokines [platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β, tumor necrosis factor (TNF)-α, interferons (IFNs), interleukins (ILs)], oxidative stress, and microRNAs (miRNAs) are involved in the initiation and progression of liver fibrosis/cirrhosis. Generally, liver fibrosis begins with the stimulation of inflammatory immune cells to secrete cytokines, growth factors, and other activator molecules. These chemical mediators direct HSCs to activate and synthesize large amounts of extracellular matrix (ECM) components. Therefore, HSC activation is a pivotal event in the development of fibrosis and a major contributor to collagen (specifically type I) accumulation. The inhibitory effect of halofuginone on collagen type α1(I) synthesis and ECM deposition has been shown in several experimental models of fibrotic diseases. Halofuginone inhibits TGF-β–induced phosphorylation of Smad3, which is a key phenomenon in the fibrogenesis. It also regulates cell growth and differentiation, apoptosis, cell migration, and immune cell function in liver fibrosis/cirrhosis. This review discusses the etiology and mechanisms of liver fibrosis/cirrhosis and the promising role of antifibrotic agent halofuginone.

**Keywords:** liver fibrosis, liver cirrhosis, hepatic stellate cells, pathogenesis, anti-fibrotic, halofuginone

## **1. Introduction**

Liver cirrhosis is the end-stage condition of several chronic liver diseases, and fibrosis is the critical pre-stage of cirrhosis. On a worldwide perspective, liver cirrhosis can be induced by

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

a number of well-defined etiological causes/factors or conditions such as chronic infection by hepatitis B, C viruses, chronic alcoholism and/or chronic exposure to toxins or drugs, infections, chronic exposure to altered metabolic conditions, inherited metabolic diseases such as hematochromatosis and Wilson's disease, auto-immune diseases such as primary biliary cirrhosis, and auto-immune hepatitis [1–3]. These etiologies may work separately or in combination with each other to produce cumulative effects. While the causes of liver cirrhosis are multifactorial, there are some pathological characteristics that are common to all cases of cirrhosis, including degeneration and necrosis of hepatocytes, replacement of healthy liver parenchyma by fibrotic scar tissues and regenerative nodules, and loss of liver function [4–7].

Fibrosis is characterized by high levels of extracellular matrix (ECM, non-functional connective tissue) components extremely rich in collagen type I. The matrix metalloproteinases (MMPs, matrix degradation enzymes), and the tissue inhibitor of metalloproteinases (TIMPs) play a crucial role in the fine regulation of ECM turnover, which is altered in most pathological states associated with liver fibrosis [8]. The key cellular mediator of fibrosis comprises the activated hepatic stellate cells (HSCs), which serve as the primary ECM-producing cells. HSCs, which play a key role in the development of liver fibrosis [9, 10], are activated by several inflammatory cytokines and growth factors in a paracrine and autocrine manner [11, 12].

Liver fibrosis and cirrhosis are dynamic and highly integrated molecular, tissue and cellular processes that can progress and regress over time [13] and that require cellular cross-talk between various liver cell types [14]. At early stages of fibrosis, initiating signals [such as DNA, reactive oxygen species (ROS)], responding cells [Kupffer cells (KCs), platelets, liver sinusoidal endothelial cells (LSECs)], and soluble mediators [such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β] induce accompanying wound-healing responses to liver injury. With time, cells, cytokine responses, and ECM components become more specialized but continue to have strong interactions with each other [15].

Halofuginone is a non-toxic plant alkaloid [7-bromo-6-chloro-3-(3-hydroxy-2-piperidine)- 2-oxopropyl-4(3H)-quinazoline] isolated from the roots of *Dichroa febrifuga*, and is used worldwide as an anti-parasitic drug [16]. Independent of this effect, halofuginone was found to be a potent inhibitor of collagen type α1 (I) gene expression [17], which was demonstrated in a broad range of cell types both *in vitro* and *in vivo* [16–20]. Due to its inhibitory effects on collagen synthesis (collagen type α1) and ECM deposition, halofuginone treatment was used in several experimental disease models characterized by excessive collagen accumulation, such as pulmonary, pancreatic and renal fibrosis [21–23], scleroderma and chronic graft-versus-host disease [24], post-operative peritendinous and abdominal adhesions [25, 26], urethral and esophageal strictures [27, 28], wound repair [29], burn injury [30], renal injury [31, 32], injury-induced arterial intimal hyperplasia [33], colitis [34], and liver fibrosis and cirrhosis [35–39]. Although the exact anti-fibrotic mechanism of halofuginone is not well understood, it was found that halofuginone affects collagen synthesis probably by inhibiting TGF-β-mediated Smad3 (intracellular protein) activation [40]. Halofuginone also regulates cell growth and differentiation, apoptosis, cell migration, and immune cell function [41]. It prevents concanavalin A-induced liver fibrosis by affecting T helper 17 (Th17) cell differentiation, which suggests a direct connection between the myofibroblasts/fibrosis pathway and the Th17 pro-inflammatory pathway [38]. In addition, halofuginone treatment effectively inhibits the delayed-type hypersensitivity response, indicating suppression of T cell–mediated inflammation *in vivo* [42]. Moreover, it is a potent inhibitor of nuclear factor (NF)-κB, pro-inflammatory cytokines and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation in activated T cells *in vitro* [42]. Also, it inhibits HSC proliferation and migration and up-regulates their expressions of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NF-κB [43].

Although there are no highly effective anti-fibrogenic agents currently available, the potential candidates that can specifically inhibit ECM components in general and specifically inhibit collagen type I in particular, are considered to be promising for the prevention and treatment of liver fibrosis/cirrhosis. The present review aims to clarify the etiology and mechanisms of liver fibrosis/cirrhosis and focus on the anti-fibrotic potential of a novel and promising agent, halofuginone.

## **2. Role of different cell types in liver fibrosis/cirrhosis**

a number of well-defined etiological causes/factors or conditions such as chronic infection by hepatitis B, C viruses, chronic alcoholism and/or chronic exposure to toxins or drugs, infections, chronic exposure to altered metabolic conditions, inherited metabolic diseases such as hematochromatosis and Wilson's disease, auto-immune diseases such as primary biliary cirrhosis, and auto-immune hepatitis [1–3]. These etiologies may work separately or in combination with each other to produce cumulative effects. While the causes of liver cirrhosis are multifactorial, there are some pathological characteristics that are common to all cases of cirrhosis, including degeneration and necrosis of hepatocytes, replacement of healthy liver parenchyma by fibrotic scar tissues and regenerative nodules, and loss of liver function [4–7]. Fibrosis is characterized by high levels of extracellular matrix (ECM, non-functional connective tissue) components extremely rich in collagen type I. The matrix metalloproteinases (MMPs, matrix degradation enzymes), and the tissue inhibitor of metalloproteinases (TIMPs) play a crucial role in the fine regulation of ECM turnover, which is altered in most pathological states associated with liver fibrosis [8]. The key cellular mediator of fibrosis comprises the activated hepatic stellate cells (HSCs), which serve as the primary ECM-producing cells. HSCs, which play a key role in the development of liver fibrosis [9, 10], are activated by several inflammatory cytokines and growth factors in a paracrine and autocrine manner [11, 12]. Liver fibrosis and cirrhosis are dynamic and highly integrated molecular, tissue and cellular processes that can progress and regress over time [13] and that require cellular cross-talk between various liver cell types [14]. At early stages of fibrosis, initiating signals [such as DNA, reactive oxygen species (ROS)], responding cells [Kupffer cells (KCs), platelets, liver sinusoidal endothelial cells (LSECs)], and soluble mediators [such as platelet-derived growth factor (PDGF), transforming growth factor (TGF)-β] induce accompanying wound-healing responses to liver injury. With time, cells, cytokine responses, and ECM components become

258 Liver Cirrhosis - Update and Current Challenges

more specialized but continue to have strong interactions with each other [15].

Halofuginone is a non-toxic plant alkaloid [7-bromo-6-chloro-3-(3-hydroxy-2-piperidine)- 2-oxopropyl-4(3H)-quinazoline] isolated from the roots of *Dichroa febrifuga*, and is used worldwide as an anti-parasitic drug [16]. Independent of this effect, halofuginone was found to be a potent inhibitor of collagen type α1 (I) gene expression [17], which was demonstrated in a broad range of cell types both *in vitro* and *in vivo* [16–20]. Due to its inhibitory effects on collagen synthesis (collagen type α1) and ECM deposition, halofuginone treatment was used in several experimental disease models characterized by excessive collagen accumulation, such as pulmonary, pancreatic and renal fibrosis [21–23], scleroderma and chronic graft-versus-host disease [24], post-operative peritendinous and abdominal adhesions [25, 26], urethral and esophageal strictures [27, 28], wound repair [29], burn injury [30], renal injury [31, 32], injury-induced arterial intimal hyperplasia [33], colitis [34], and liver fibrosis and cirrhosis [35–39]. Although the exact anti-fibrotic mechanism of halofuginone is not well understood, it was found that halofuginone affects collagen synthesis probably by inhibiting TGF-β-mediated Smad3 (intracellular protein) activation [40]. Halofuginone also regulates cell growth and differentiation, apoptosis, cell migration, and immune cell function [41]. It prevents concanavalin A-induced liver fibrosis by affecting T helper 17 (Th17) cell differentiation, which suggests a direct connection between the myofibroblasts/fibrosis pathway and The liver is composed of parenchymal cells (hepatocytes) and non-parenchymal cells (HSCs, LSECs, and KCs). Both parenchymal and non-parenchymal cells are involved in the initiation and progression of liver fibrosis/cirrhosis (**Table 1**).


*Abbreviations*: TGF-β, transforming growth factor-β; IL, interleukin; TRAIL, tumor necrosis factor-related apoptosisinducing ligand.

**Table 1.** Role of different cell types in liver fibrosis/cirrhosis.

#### **2.1. Hepatic stellate cells (HSCs)**

HSCs are one of the non-parenchymal cells of the liver located in the perisinusoidal space (space of Disse) between hepatocytes and sinusoidal endothelial cells. HSCs are also known as fat-storing cells, perisinusoidal cells, lipocytes, or vitamin A-rich cells, and their main function is storage of vitamin A and other retinoids [7, 44]. HSCs show two different phenotypes: quiescent type in the healthy liver and activated type in the diseased one. Quiescent HSCs mostly function as vitamin A reserves [45]. However, in response to liver injury, inflammatory cytokines such as tumor necrosis factor (TNF)-α, TGF-β, interleukin (IL)-1, and PDGF promote HSCs to undergo a phenotypic switch from a quiescent, vitamin A storing cell into proliferative, α-smooth muscle actin (α-SMA)-positive, myofibroblast-like cells which contribute to fibrosis by producing the abnormal ECM components [46]. Therefore, HSC activation is a pivotal phenomenon in initiation and progression of liver fibrosis and a major contributor to collagen accumulation [47, 48].

#### **2.2. Hepatocytes**

Hepatocytes are the primary parenchymal component of the liver and play an important role in fibrosis/cirrhosis. They are the main targets of several hepatotoxic agents including hepatitis viruses, alcohol metabolites, and bile acids [11]. Liver injury either promotes apoptosis or triggers compensatory regeneration of hepatocytes [49]. Hepatocyte-derived apoptotic bodies stimulate secretion of fibrogenic cytokines from KCs and promote HSC activation via interaction of toll-like receptor (TLR)-9 with DNA, which is released from apoptotic hepatocytes [50–53]. On the other hand, activated HSCs also act as phagocytes and phagocytize hepatocyte apoptotic bodies, which promote myofibroblasts survival and fibrogenesis [54]. Therefore, apoptosis of hepatocytes is a crucial event in liver injury and contributes to tissue inflammation, fibrogenesis, and development of cirrhosis. Also, in the cirrhotic stage, hypoxic hepatocytes become a primary source of TGF-β, which may augment liver fibrosis [55].

#### **2.3. Liver sinusoidal endothelial cells (LSECs)**

LSECs constitute the sinusoidal wall, also known as endothelium, or endothelial lining. The main characteristic of LSECs is having the fenestrae on the surface of the endothelium [56, 57]. The endothelial fenestrae control exchange of fluids, solutes, and particles between sinusoidal blood and hepatocytes [58]. In the healthy liver, the fenestrated endothelial cells prevent HSC activation through vascular endothelial growth factor-stimulated nitric oxide production [59]. However, LSECs have high endocytotic capacity [56, 60]. Upon liver injury, defenestration and capillarization of LSECs lead to impaired substrate exchange which is the major cause of hepatic dysfunction [57, 58] and HSC activation [61, 62]. It has been also revealed that LSECs can secrete the cytokine IL-33 to activate HSCs and promote liver fibrosis [63].

#### **2.4. Kupffer cells (KCs)**

KCs, also called stellate macrophages, are interspersed throughout the liver, situated within the sinusoids. KCs are responsible for the removal of circulating microorganisms, immune complexes, and debris from the blood stream. They are usually activated by many injurious factors such as viral infection and alcohol [64]. Activation of KCs is a key phenomenon in initiation and preservation of liver fibrosis. Activated KCs express chemokine receptors, secret inflammatory cytokines (such as TNF-α, IL-1, IL-6) and serve as antigen-presenting cells, which lead to progression of fibrosis [65–68]. KCs are also involved in the activation of HSCs and formation of liver fibrosis. For example, KC-conditioned medium promotes activation of cultured rat HSCs with enhanced ECM production and stimulates cell proliferation via induction of PDGF receptors on the membrane of HSCs [69]. KC-derived TGF-β1 stimulates proliferation and collagen formation of HSCs in a rat model of alcoholic liver fibrogenesis [66]. Moreover, macrophage ablation has been shown to attenuate liver fibrosis. For example, gadolinium chloride-mediated depletion of KCs has been shown to result in attenuation of carbon tetrachloride (CCl<sup>4</sup> )-induced fibrosis in rats with prevention of the increased TGF-β expression [70]. Conversely, KCs produce interstitial collagenase MMP-13 when treated with gadolinium chloride, which reduces ECM deposition during experimental liver fibrosis [71]. In addition, activated KCs can effectively kill HSCs by a caspase 9-dependent mechanism via possible involvement of TNF-related apoptosis-inducing ligand (TRAIL) [72, 73].

## **3. Role of cytokines in liver fibrosis/cirrhosis**

Cytokines, which mediate several immune and inflammatory reactions, are small signaling proteins that facilitate intercellular communication between various cells. They function through cell-surface receptors, and down-stream signaling induces an alteration of cell functions. Liver fibrosis/cirrhosis is a result of interaction of a complex network of cytokines, which modify activities of circulating immune cells, HSCs, KCs, LSECs, and hepatocytes. The role of cytokines in liver fibrosis/cirrhosis is summarized in **Table 2**.

#### **3.1. Platelet-derived growth factor (PDGF)**

**2.1. Hepatic stellate cells (HSCs)**

260 Liver Cirrhosis - Update and Current Challenges

contributor to collagen accumulation [47, 48].

**2.3. Liver sinusoidal endothelial cells (LSECs)**

**2.4. Kupffer cells (KCs)**

**2.2. Hepatocytes**

HSCs are one of the non-parenchymal cells of the liver located in the perisinusoidal space (space of Disse) between hepatocytes and sinusoidal endothelial cells. HSCs are also known as fat-storing cells, perisinusoidal cells, lipocytes, or vitamin A-rich cells, and their main function is storage of vitamin A and other retinoids [7, 44]. HSCs show two different phenotypes: quiescent type in the healthy liver and activated type in the diseased one. Quiescent HSCs mostly function as vitamin A reserves [45]. However, in response to liver injury, inflammatory cytokines such as tumor necrosis factor (TNF)-α, TGF-β, interleukin (IL)-1, and PDGF promote HSCs to undergo a phenotypic switch from a quiescent, vitamin A storing cell into proliferative, α-smooth muscle actin (α-SMA)-positive, myofibroblast-like cells which contribute to fibrosis by producing the abnormal ECM components [46]. Therefore, HSC activation is a pivotal phenomenon in initiation and progression of liver fibrosis and a major

Hepatocytes are the primary parenchymal component of the liver and play an important role in fibrosis/cirrhosis. They are the main targets of several hepatotoxic agents including hepatitis viruses, alcohol metabolites, and bile acids [11]. Liver injury either promotes apoptosis or triggers compensatory regeneration of hepatocytes [49]. Hepatocyte-derived apoptotic bodies stimulate secretion of fibrogenic cytokines from KCs and promote HSC activation via interaction of toll-like receptor (TLR)-9 with DNA, which is released from apoptotic hepatocytes [50–53]. On the other hand, activated HSCs also act as phagocytes and phagocytize hepatocyte apoptotic bodies, which promote myofibroblasts survival and fibrogenesis [54]. Therefore, apoptosis of hepatocytes is a crucial event in liver injury and contributes to tissue inflammation, fibrogenesis, and development of cirrhosis. Also, in the cirrhotic stage, hypoxic hepatocytes become a primary source of TGF-β, which may augment liver fibrosis [55].

LSECs constitute the sinusoidal wall, also known as endothelium, or endothelial lining. The main characteristic of LSECs is having the fenestrae on the surface of the endothelium [56, 57]. The endothelial fenestrae control exchange of fluids, solutes, and particles between sinusoidal blood and hepatocytes [58]. In the healthy liver, the fenestrated endothelial cells prevent HSC activation through vascular endothelial growth factor-stimulated nitric oxide production [59]. However, LSECs have high endocytotic capacity [56, 60]. Upon liver injury, defenestration and capillarization of LSECs lead to impaired substrate exchange which is the major cause of hepatic dysfunction [57, 58] and HSC activation [61, 62]. It has been also revealed that LSECs

KCs, also called stellate macrophages, are interspersed throughout the liver, situated within the sinusoids. KCs are responsible for the removal of circulating microorganisms, immune

can secrete the cytokine IL-33 to activate HSCs and promote liver fibrosis [63].

PDGF is one of the most potent mitogen for HSCs isolated from mouse, rat, or human liver [74]. PDGF and its receptors are significantly overexpressed in fibrotic tissues, and its activity increases with the degree of liver fibrosis [75, 76]. Hepatocyte damage resulting from factors, such as viruses, chemicals, or hepatotoxins, can induce KCs to synthesize and release PDGF [77]. When PDGF binds to its specific receptor on the membrane of HSCs, it activates corresponding signal molecules and transcription factors, leading to the activation of its downstream target genes and activation of HSCs [74]. PDGF has been shown to up-regulate the expression of MMP-2, MMP-9, and TIMP-1, and inhibit collagenase activity, thereby decreasing ECM degradation [78].

#### **3.2. Transforming growth factor (TGF)-β**

Among fibrotic mediators, TGF-β is one of the most important pro-fibrotic cytokine. The direct targets in TGF-β pathway, Smads (especially Smad3) are critical mediators in fibrogenesis [79, 80]. The intracellular effectors of TGF-β signaling, the Smad proteins, are activated by receptors and


*Abbreviations*: HSC, hepatic stellate cell; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; ECM, extracellular matrix; SMA, smooth muscle actin; MCP, monocyte chemoattractant protein; TLR, toll-like receptor; KC, Kupffer cell; STAT, signal transducer and activator of transcription; NF-κB, nuclear factor-κB.

**Table 2.** Role of cytokines in liver fibrosis/cirrhosis.

translocate into the nucleus, where they regulate transcription [79]. The main effect of TGF-β is to stimulate HSC activation, and the TGF-β autocrine cycle in activated HSCs is an important positive feedback to the progression of liver fibrosis [81, 82]. Though the main source of TGF-β in fibrotic liver is activated HSCs, LSECs, KCs, and hepatocytes also contribute to synthesis of this growth factor [83]. The level of TGF-β1 expression is increased during liver fibrosis and reaches a maximum at cirrhosis [55]. TGF-β1 induces expression of the matrix-producing genes, inhibits ECM degradation, and promotes TIMPs, leading to excessive collagen accumulation and promoting the development of liver fibrosis [84, 85]. Furthermore, TGF-β1 has been shown to inhibit DNA synthesis and induces apoptosis of hepatocytes. In particular, TGF-β1-induced apoptosis is thought to be responsible for tissue loss and decrease in liver size seen in cirrhosis [86–88].

#### **3.3. Tumor necrosis factor (TNF)-α**

**Mediators Mechanism of action References**

inhibits collagenase activity

degradation, and promotes TIMPs

Tumor necrosis factor (TNF)-α Induces hepatocyte death by apoptosis [90]

IFN-α Triggers apoptosis of HSCs [96]

IFN-γ Reduces ECM deposition by inhibiting HSC activation [98]

IFN-β Decreases α-SMA and collagen expression and inhibits HSC

IL-1 Activates HSCs and stimulates them to produce MMP-9, MMP-

IL-17 Regulates production of TGF-β1 by KCs, induces activation of

IL-6 Attenuates hepatocyte apoptosis and induces regeneration of hepatocytes through NF-κB pathway

KC, Kupffer cell; STAT, signal transducer and activator of transcription; NF-κB, nuclear factor-κB.

IL-10 Inhibits expression of TGF-β1, MMP-2 and TIMP-1 [115]

IL-22 Inhibits hepatocyte apoptosis via STAT3 [121, 122]

*Abbreviations*: HSC, hepatic stellate cell; MMP, matrix metalloproteinase; TIMP, tissue inhibitor of metalloproteinase; ECM, extracellular matrix; SMA, smooth muscle actin; MCP, monocyte chemoattractant protein; TLR, toll-like receptor;

via STAT3 pathway

13 and TIMP-1

macrophages

up-regulation

**Table 2.** Role of cytokines in liver fibrosis/cirrhosis.

expression

Activates HSCs [74]

Stimulates HSC activation [81, 82]

Inhibits DNA synthesis and induces apoptosis of hepatocytes [86–88]

Activates HSCs and stimulates ECM synthesis [91, 92] Induces/reduces apoptosis of activated HSCs [73, 93]

Elicits an anti-apoptotic effect on activated HSCs [100]

Exerts a pro-apoptotic effect on activated HSCs [100]

[78]

[84, 85]

[94]

[97]

[102]

[105]

[108]

[112]

[120]

Up-regulates expression of MMP-2, MMP-9, and TIMP-1 and

Induces expression of matrix-producing genes, inhibits ECM

Reduces glutathione and inhibits pro-collagen α1 mRNA

activation through inhibition of TGF-β and PDGF

Increases MCP-1 in hepatocytes and augments TLR-4 dependent up-regulation of inflammatory signaling in

HSCs and induces production of collagen and α-SMA in HSCs

Inhibits HSC activity [117]

Induces HSC senescence [123]

Reduces TGF-β1, TNF-α, collagen α1, and TIMP mRNA

Platelet-derived growth factor

262 Liver Cirrhosis - Update and Current Challenges

Transforming growth factor

(PDGF)

(TGF)-β

Interferons (IFNs)

Interleukins (ILs)

TNF-α is a pro-inflammatory cytokine produced by different cell types. However, it is mainly produced by activated KCs in the liver. TNF-α is an important mediator in several processes such as cell proliferation, inflammation, and apoptosis [89]. TNF-α can induce cell death by apoptosis, and KCs can be stimulated by apoptotic hepatocytes to produce more TNF-α [90]. Furthermore, TNF-α plays an essential role in the HSC activation and ECM synthesis in liver fibrosis [91, 92]. TNF-α may act as surviving factor for activated rat HSCs by up-regulating the anti-apoptotic factors (NF-κB, bcl-xL, and p21WAF1) and by down-regulating the proapoptotic factor (p53) [93]. On the other hand, TNF-α can induce apoptosis in HSCs [73]. It has been also demonstrated that TNF-α shows anti-fibrogenic effect in rat HSCs by reducing glutathione and inhibiting pro-collagen α1 mRNA expression [94].

#### **3.4. Interferons (IFNs)**

IFNs are potent pleiotropic cytokines that broadly alter cellular functions in response to viral and other infections. Leukocytes synthesize IFN-α and IFN-β in response to viruses, and T cells secrete IFN-γ upon stimulation with various antigens and mitogens. Although the primary action of IFN-α is to eradicate viruses, patients with hepatitis C treated with IFN-α exhibit a regression of liver fibrosis even if viral eradication is not achieved [95], indicating that IFN-α itself has anti-fibrotic activity via triggering the apoptosis of HSCs [96]. IFN-β treatment decreases α-SMA and collagen expression and inhibits HSC activation through inhibition of TGF-β and PDGF pathways [97]. Similarly, IFN-γ reduces ECM deposition *in vivo* by inhibiting HSC activation [98] via TGFβ1/Smad3 signaling pathways [99]. Interestingly, IFN-α and IFN-γ may exert opposite effects on apoptosis in HSCs. IFN-α was shown to elicit an anti-apoptotic effect on activated HSCs, whereas IFN-γ was found to exert pro-apoptotic effect on HSCs by down-regulating heat-shock protein 70 [100].

#### **3.5. Interleukins (ILs)**

ILs are immunomodulatory cytokines that are critically involved in the regulation of immune responses. They are produced by a variety of cell types such as CD4<sup>+</sup> T lymphocytes, monocytes, macrophages, and endothelial cells. KCs and LSECs can rapidly produce ILs in response to liver injury. ILs can have pro- and anti-inflammatory functions in chronic liver diseases, dependent on the inflammatory stimulus and, the producing and the responding cell type.

The main function of pro-inflammatory ILs is to stimulate immune responses that result in the elimination of invading pathogens or damaged cells. On the other hand, anti-inflammatory ILs are produced to protect the host's body from exaggerated immune responses and to limit organ damage. As soon as the pathogenic stimuli are removed, ILs production is no longer needed, and inflammation diminishes. If the stimulus continues, inflammation can become chronic and induce a variety of inflammatory diseases [101].

IL-1 is a pro-inflammatory and pro-fibrotic cytokine that directly activates HSCs and stimulates them to produce MMP-9, MMP-13, and TIMP-1, resulting in liver fibrogenesis [102]. IL-1 receptor-deficient mice exhibits ameliorated liver damage and reduced fibrogenesis [102]. Similarly, IL-1 receptor antagonist protects rats from developing fibrosis in dimethylnitrosamine-induced liver fibrosis [103]. Lack of IL-1α or IL-1β also makes the mice less susceptible to develop liver fibrosis in experimental model of steatohepatitis [104]. It has been also shown that IL-1β at physiological doses increases the inflammatory and prosteatotic chemokine monocyte chemoattractant protein (MCP)-1 in hepatocytes, and augments TLR-4-dependent up-regulation of inflammatory signaling in macrophages [105]. Thus, IL-1 is an important participant, along with other cytokines, and controls the progression from liver injury to fibrogenesis.

Another pro-inflammatory and pro-fibrotic cytokine IL-17 has been reported to be involved in many immune processes, most notably in inducing and mediating pro-inflammatory responses. Its expression increases with increasing degree of liver fibrosis [106, 107], suggesting that IL-17 may not only induce inflammation but also contribute to disease progression and chronicity [106]. IL-17 regulates production of TGF-β1 by KCs, which in turn, induces activation of HSCs into myofibroblasts, and further facilitates differentiation of IL-17 expressing cells [108]. Also, IL-17 directly induces production of collagen and α-SMA in HSCs via the signal transducer and activator of transcription (STAT)3 signaling pathway [108]. Furthermore, abrogation of IL-17 signaling by deletion of IL-17RA protects mice from fibrogenesis [108]. Similarly, blockade of endogenous IL-17 with neutralizing IL-17-specific antibody reduces liver fibrosis, whereas treatment with recombinant IL-17 increases fibrosis development [109].

IL-6 is a pleiotropic cytokine, which may affect differentiation of fibroblast to myofibroblast, and it plays an important role in fibrotic diseases [110, 111]. On the other hand, IL-6 has beneficial effects for the liver. For example, IL-6 reduces CCl<sup>4</sup> -induced acute and chronic liver injury and fibrosis [112]. Also, it attenuates hepatocyte apoptosis and induces regeneration of hepatocytes through NF-κB signaling pathway [112]. In an experimental model of concavaline A-induced hepatitis, IL-6 pretreatment protects mice from liver injury. This protection requires gp130 signaling in hepatocytes and is mediated via the gp130/STAT3 signaling cascade [113]. Furthermore, systemic injection of IL-6 followed by intrahepatic transplantation of mesenchymal stem cells is also able to reduce hepatocyte apoptosis and liver fibrogenesis after CCl<sup>4</sup> treatment [114].

IL-10 is one of the major anti-inflammatory cytokines, with tissue protective functions during fibrogenesis. It down-regulates the pro-inflammatory response and has a modulatory effect on liver fibrogenesis [115, 116]. IL-10 has been shown to exert anti-fibrotic effects through inhibiting HSC activity [117]. IL-10-deficient mice show higher liver fibrosis with larger inflammatory infiltrates in CCl<sup>4</sup> -induced liver fibrosis compared to wild-type mice [118, 119]. IL-10 gene therapy reverses CCl<sup>4</sup> -induced murine liver fibrosis by inhibiting the expression of TGF-β1, MMP-2, and TIMP-1 [115]. Additionally, IL-10 gene therapy reverses liver fibrosis and prevents cell apoptosis in a thioacetamide-treated murine liver, and reduces TGF-β1, TNF-α, collagen α1, and TIMP mRNA up-regulation, suggesting a therapeutic potential for treatment with IL-10 [120].

IL-22 is known to play important roles in the modulation of tissue immune responses to inflammation. It reduces inflammation-induced damage of hepatocytes both *in vitro* and *in vivo* by promoting their survival and inhibiting apoptosis [121]. This protective function is dependent on STAT3 signaling, as STAT3-deficient mice were not protected when treated with IL-22 [122]. Similarly, in CCl<sup>4</sup> -induced liver fibrogenesis, IL-22 is protective through induction of senescence in HSCs via STAT3 signaling pathway [123]. Moreover, IL-22 is also involved in the restoration of functional liver mass after organ damage. Liver progenitor cells have been shown to express IL-22R, and IL-22 derived from inflammatory cells induces proliferation of liver progenitor cells [124].

## **4. Role of oxidative stress in liver fibrogenesis**

The main function of pro-inflammatory ILs is to stimulate immune responses that result in the elimination of invading pathogens or damaged cells. On the other hand, anti-inflammatory ILs are produced to protect the host's body from exaggerated immune responses and to limit organ damage. As soon as the pathogenic stimuli are removed, ILs production is no longer needed, and inflammation diminishes. If the stimulus continues, inflammation can become

IL-1 is a pro-inflammatory and pro-fibrotic cytokine that directly activates HSCs and stimulates them to produce MMP-9, MMP-13, and TIMP-1, resulting in liver fibrogenesis [102]. IL-1 receptor-deficient mice exhibits ameliorated liver damage and reduced fibrogenesis [102]. Similarly, IL-1 receptor antagonist protects rats from developing fibrosis in dimethylnitrosamine-induced liver fibrosis [103]. Lack of IL-1α or IL-1β also makes the mice less susceptible to develop liver fibrosis in experimental model of steatohepatitis [104]. It has been also shown that IL-1β at physiological doses increases the inflammatory and prosteatotic chemokine monocyte chemoattractant protein (MCP)-1 in hepatocytes, and augments TLR-4-dependent up-regulation of inflammatory signaling in macrophages [105]. Thus, IL-1 is an important participant, along with other cytokines, and controls the progression from liver injury to

Another pro-inflammatory and pro-fibrotic cytokine IL-17 has been reported to be involved in many immune processes, most notably in inducing and mediating pro-inflammatory responses. Its expression increases with increasing degree of liver fibrosis [106, 107], suggesting that IL-17 may not only induce inflammation but also contribute to disease progression and chronicity [106]. IL-17 regulates production of TGF-β1 by KCs, which in turn, induces activation of HSCs into myofibroblasts, and further facilitates differentiation of IL-17 expressing cells [108]. Also, IL-17 directly induces production of collagen and α-SMA in HSCs via the signal transducer and activator of transcription (STAT)3 signaling pathway [108]. Furthermore, abrogation of IL-17 signaling by deletion of IL-17RA protects mice from fibrogenesis [108]. Similarly, blockade of endogenous IL-17 with neutralizing IL-17-specific antibody reduces liver fibrosis, whereas treatment with recombinant IL-17 increases fibrosis development [109]. IL-6 is a pleiotropic cytokine, which may affect differentiation of fibroblast to myofibroblast, and it plays an important role in fibrotic diseases [110, 111]. On the other hand, IL-6 has ben-

injury and fibrosis [112]. Also, it attenuates hepatocyte apoptosis and induces regeneration of hepatocytes through NF-κB signaling pathway [112]. In an experimental model of concavaline A-induced hepatitis, IL-6 pretreatment protects mice from liver injury. This protection requires gp130 signaling in hepatocytes and is mediated via the gp130/STAT3 signaling cascade [113]. Furthermore, systemic injection of IL-6 followed by intrahepatic transplantation of mesenchymal stem cells is also able to reduce hepatocyte apoptosis and liver fibrogenesis

IL-10 is one of the major anti-inflammatory cytokines, with tissue protective functions during fibrogenesis. It down-regulates the pro-inflammatory response and has a modulatory effect on liver fibrogenesis [115, 116]. IL-10 has been shown to exert anti-fibrotic effects through inhibiting HSC activity [117]. IL-10-deficient mice show higher liver fibrosis with larger


chronic and induce a variety of inflammatory diseases [101].

264 Liver Cirrhosis - Update and Current Challenges

eficial effects for the liver. For example, IL-6 reduces CCl<sup>4</sup>

fibrogenesis.

after CCl<sup>4</sup>

treatment [114].

Oxidative stress is caused by an imbalance between production of ROS and their elimination by anti-oxidant defenses [125]. As liver is an essential organ for detoxification and nutrients metabolism, it is more vulnerable to oxidative stress [125]. Oxidative stress-related molecules and pathways modulate tissue and cellular events involved in the liver fibrogenesis [126]. The generation of ROS plays a crucial role in producing liver damage and initiating liver fibrogenesis [126]. Oxidative stress disrupts lipids, proteins and DNA, induces necrosis and apoptosis of hepatocytes, resulting in the initiation of fibrosis [127]. ROS stimulate the production of pro-fibrogenic mediators from KCs and circulating inflammatory cells. Remarkably, ROS directly activate HSCs. The elevated oxidative stress contributes to fibrogenesis via stimulating collagen production from activated HSCs and release of other pro-fibrogenic cytokines and growth factors [126, 128].

## **5. Role of microRNAs (miRNAs) in liver pathophysiology**

miRNAs are a family of small non-coding RNAs (20–25 nucleotides in length) that control gene expression by binding to mRNAs to repress translation or induce mRNA cleavage [129]. Many researchers have reported that the unusual expression of miRNAs in liver tissue was related to the pathogenesis of liver disease of any etiology [130, 131]. Recently, miRNAs have been found to play fundamental roles in liver fibrosis, including those in HSC activation and ECM production [132]. For example, miRNA-21 exhibits an important role in the pathogenesis and progression of liver fibrosis. A natural product 3,3′-Diindolylmethane (DIM) inhibits TGF-β signaling pathway by down-regulating the miRNA-21 expression in thioacetamide-induced experimental liver fibrosis. Furthermore, DIM can suppress HSC activation via down-regulating

miRNA-21 levels in HSCs by inhibiting activity of the transcription factor AP-1 [133]. Inhibition of miRNA-21 also reduces liver fibrosis through concomitant reduction of CD24<sup>+</sup> liver progenitor cells [134]. In mouse and human studies, the expression levels of miRNA-199a, antisense miRNA-199a\*, miRNA-200a, and miRNA-200b are found to be positively and significantly correlated with progression of liver fibrosis. Overexpression of these miRNAs dramatically increases the expression of fibrosis-related genes in HSCs [135]. Also, miRNA-221 and miRNA-222 are up-regulated in human liver in a fibrosis progression-dependent manner [136]. Similarly, in isolated primary human liver cells, miRNA-571 is up-regulated in hepatocytes and HSCs in response to the pro-fibrogenic cytokine TGF-β [137]. miRNA-214 appears to participate in the development of liver fibrosis by modulating the epidermal growth factor (EGF) receptor and TGF-β signaling pathways. Also, inhibition of miRNA-214 by locked nucleic acid-antimiRNA-214 ameliorates liver fibrosis in PDGF c transgenic mice [138]. In addition, miRNA-214-5p may play crucial roles in HSC activation and progression of liver fibrosis. The overexpression of miRNA-214-5p in human stellate cells increases the expression of fibrosis-related genes such as MMP-2, MMP-9, α-SMA, and TGF-β1 [139].

miRNAs may also play anti-fibrogenic roles. It has been demonstrated that both miRNA-150 and miRNA-194 inhibit HSC activation and ECM production in rats with liver fibrosis by decreasing the expression of c-myb (target for miRNA-150) and rac 1 (target for miRNA-194) [140]. Interestingly, miRNAs such as miRNA-19b, miRNA-29, miRNA-133a, and miRNA-146a are significantly down-regulated in HSCs isolated from experimental animals with liver fibrosis, and restoration of these miRNAs alleviates fibrogenesis [47, 141, 142]. Moreover, miRNA-133a overexpression inhibits both human and murine primary HSCs proliferation and prevents the progression of liver fibrosis [142].

Multiple studies have proposed that miRNAs may serve as biomarkers for HSC activation and liver fibrosis progression, and can be possible candidates for future therapies targeting liver fibrosis/cirrhosis.

## **6. Pathogenesis of liver fibrosis/cirrhosis**

Liver fibrosis and its end-stage consequence, cirrhosis, represent the final common pathway of almost all chronic liver diseases. Fibrosis and cirrhosis of the liver remain major medical problems with significant morbidity and mortality worldwide. Liver fibrosis is in fact a wound-healing response to liver injury and is characterized by accumulation of fibrotic scar tissue. Although the scar tissue formation is beneficial at first because it encapsulates the injury, the chronic activation of this healing process eventually progresses to advanced fibrosis/cirrhosis. This leads to altered vascular architecture and microcirculation, ischemia, and widespread hepatocyte cell death [143]. Also, in cirrhosis, collagen strands become so prevalent and divide the liver parenchyma into distinct structurally abnormal regenerative nodules, resulting in organ dysfunction [143].

In fact, liver damage leading to cirrhosis is the result of a complex mechanism involving, from direct toxic effects to a sustained inflammatory process, driving to the death of hepatocytes via apoptosis and liver fibrosis, mediated by secretion of several cytokines [144]. The inflammatory reaction is the coordinated process by which the liver responds to local insults, trying to restore the hepatic architecture and function after acute liver injury [128]. However, if the liver is faced to a sustained local damage, the chronic inflammatory response gives rise to a progressive replacement of healthy liver tissue by non-functional fibrotic scar tissue. The imbalance between tissue regeneration and fibrosis determines the outcome toward health recovery or liver cirrhosis [144].

#### **6.1. Imbalance between extracellular matrix synthesis and degradation**

miRNA-21 levels in HSCs by inhibiting activity of the transcription factor AP-1 [133]. Inhibition of miRNA-21 also reduces liver fibrosis through concomitant reduction of CD24<sup>+</sup> liver progenitor cells [134]. In mouse and human studies, the expression levels of miRNA-199a, antisense miRNA-199a\*, miRNA-200a, and miRNA-200b are found to be positively and significantly correlated with progression of liver fibrosis. Overexpression of these miRNAs dramatically increases the expression of fibrosis-related genes in HSCs [135]. Also, miRNA-221 and miRNA-222 are up-regulated in human liver in a fibrosis progression-dependent manner [136]. Similarly, in isolated primary human liver cells, miRNA-571 is up-regulated in hepatocytes and HSCs in response to the pro-fibrogenic cytokine TGF-β [137]. miRNA-214 appears to participate in the development of liver fibrosis by modulating the epidermal growth factor (EGF) receptor and TGF-β signaling pathways. Also, inhibition of miRNA-214 by locked nucleic acid-antimiRNA-214 ameliorates liver fibrosis in PDGF c transgenic mice [138]. In addition, miRNA-214-5p may play crucial roles in HSC activation and progression of liver fibrosis. The overexpression of miRNA-214-5p in human stellate cells increases the expression of fibrosis-related genes such as MMP-2, MMP-9, α-SMA, and TGF-β1 [139].

miRNAs may also play anti-fibrogenic roles. It has been demonstrated that both miRNA-150 and miRNA-194 inhibit HSC activation and ECM production in rats with liver fibrosis by decreasing the expression of c-myb (target for miRNA-150) and rac 1 (target for miRNA-194) [140]. Interestingly, miRNAs such as miRNA-19b, miRNA-29, miRNA-133a, and miRNA-146a are significantly down-regulated in HSCs isolated from experimental animals with liver fibrosis, and restoration of these miRNAs alleviates fibrogenesis [47, 141, 142]. Moreover, miRNA-133a overexpression inhibits both human and murine primary HSCs proliferation

Multiple studies have proposed that miRNAs may serve as biomarkers for HSC activation and liver fibrosis progression, and can be possible candidates for future therapies targeting

Liver fibrosis and its end-stage consequence, cirrhosis, represent the final common pathway of almost all chronic liver diseases. Fibrosis and cirrhosis of the liver remain major medical problems with significant morbidity and mortality worldwide. Liver fibrosis is in fact a wound-healing response to liver injury and is characterized by accumulation of fibrotic scar tissue. Although the scar tissue formation is beneficial at first because it encapsulates the injury, the chronic activation of this healing process eventually progresses to advanced fibrosis/cirrhosis. This leads to altered vascular architecture and microcirculation, ischemia, and widespread hepatocyte cell death [143]. Also, in cirrhosis, collagen strands become so prevalent and divide the liver parenchyma into distinct structurally abnormal regenerative

In fact, liver damage leading to cirrhosis is the result of a complex mechanism involving, from direct toxic effects to a sustained inflammatory process, driving to the death of hepatocytes

and prevents the progression of liver fibrosis [142].

**6. Pathogenesis of liver fibrosis/cirrhosis**

nodules, resulting in organ dysfunction [143].

liver fibrosis/cirrhosis.

266 Liver Cirrhosis - Update and Current Challenges

Liver fibrosis can be defined as a dynamic and highly integrated molecular, tissue and cellular process regarded as the result of an imbalance between ECM synthesis and degradation. In the healthy liver, ECM is composed of several components such as collagens (mainly the interstitial types I, III, V, VI, and the basement membrane types IV, XV, XVIII, and XIX), glycoproteins (such as laminin isoforms and fibronectin), proteoglycans and elastin [145–147]. Normally, ECM components comprise less than 3% of the relative area of a liver tissue section and approximately 0.5% of the wet weight. During the development of liver fibrosis, there is a 5- to 10-fold increase in the content of collagenous and non-collagenous components, particularly of fibrillar collagen type I and III [146], and an increase of elastin, laminins, and proteoglycans [148]. The total amount of ECM is not only dependent on the rate of synthesis but also largely on the balance between the matrix MMPs, and the TIMPs, especially TIMP-1 [15].

The MMPs are a family of zinc-dependent endopeptidases that can degrade both collagenous and non-collagenous components of ECM in the extracellular space [149]. MMP activity is regulated by TIMPs, which bind to MMPs, blocking their proteolytic activity. The MMPs and TIMPs play a crucial role in the fine regulation of the ECM turnover and the resulting increase in the TIMPs/MMPs ratio in liver promotes fibrosis by protecting accumulated matrix from degradation by MMPs (**Figure 1**) [8].

#### **6.2. Mechanisms and mediators of liver fibrogenesis**

Liver fibrosis, which is characterized by the excessive deposition of ECM (non-functional connective tissue) components [150], involves both parenchymal and non-parenchymal cells, as well as infiltrating immune cells [151, 152]. Furthermore, several critical signaling pathways have important roles in liver fibrosis. The complex interactions between these signaling pathways and different cells contribute to the progression of liver fibrosis [153].

HSCs are central effectors of fibrogenesis although other cells and processes can make significant contributions. In the healthy liver, HSCs are in a quiescent state with low proliferation rates, store dietary vitamin A, control the ECM synthesis, regulate the local vascular contractility, and serve as the pericytes for the sinusoidal endothelial cells. Damage to hepatocytes activates HSCs transformation into myofibroblast-like cells that play a fundamental role in the development of fibrotic liver response [14]. Myofibroblast-like cells with high proliferative capacity, without vitamin A, exhibit increased expression of α-SMA fibers [3]. These cells contribute to fibrosis by producing large amounts of ECM components and collagens (specifically type I) to encapsulate

**Figure 1.** Imbalances in ECM synthesis and degradation result in liver fibrosis. Regulation of degradation is determined by the balance between the activity of MMPs and TIMPs. The MMPs degrade both collagenous and non-collagenous components of ECM in the extracellular space. MMP activity is regulated by TIMPs, which bind to MMPs, blocking their proteolytic activity. Increase in the TIMPs/MMPs ratio in liver promotes fibrosis by protecting accumulated matrix from degradation by MMPs. ECM, extracellular matrix; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitor of metalloproteinases.

the injury [152]. Although HSCs are classically considered to be a major source of myofibroblasts [154, 155], other cell types like portal myofibroblasts and cells recruited from the bone marrow also contribute to the expansion of the myofibroblast population observed during the liver injury [154]. Activated HSCs also secrete an increased amount of MMPs and their inhibitors, TIMPs, which are necessary for the ECM remodeling [154, 156]. HSC activation leads to the up-regulation of TIMPs and TGF-β1 with the inhibition of MMP activity. The TIMP activation thus stimulates collagen type I synthesis and ECM deposition in the extracellular space [157]. Besides injured hepatocytes, hepatic macrophages (KCs), endothelial cells, and lymphocytes also drive HSC activation [158].

HSC activation is still the primary pathway leading to the liver fibrosis and it consists of two main stages: initiation and perpetuation (**Figure 2**) [126]. The initiation stage is related with the early changes in gene expression and phenotype that render the cells responsive to several cytokines and stimuli. Initiation of HSC activation is stimulated by several soluble factors such as oxidant stress signals (ROS), apoptotic bodies, and paracrine stimuli from neighboring cell types including hepatocytes, KCs, sinusoidal endothelium, and platelets [8, 72]. Hepatocytes The Promising Role of Anti-Fibrotic Agent Halofuginone in Liver Fibrosis/Cirrhosis http://dx.doi.org/10.5772/intechopen.68641 269

**Figure 2.** Initiation, perpetuation, and regression of liver fibrogenesis involving HSCs. The pathways of HSC activation consist of initiation and perpetuation. Initiation is stimulated by soluble factors such as apoptotic bodies, oxidant stress signals (ROS), and paracrine stimuli from neighboring cell types. Perpetuation includes HSC activation (phenotypic switch from a quiescent type into an activated type) and related cellular changes such as proliferation, chemotaxis, fibrogenesis, contractility, and abnormal matrix degradation. Repetitive damage to liver causes perpetuation of activated HSCs in the liver. Activated HSCs produce excessive collagen, down-regulate release of MMPs and enhance expression of the physiological inhibitors of the MMPs (TIMPs). Imbalances in collagen synthesis and degradation result in liver fibrosis/cirrhosis. During regression, activated HSCs undergo apoptosis or inactivation if the cause of liver injury is removed. ROS, reactive oxygen species; KC, Kupffer cell; HSC, hepatic stellate cell; TNF-α, tumor necrosis factor-α; IL, interleukin; MCP-1, monocyte chemoattractant protein-1; TGF-β, transforming growth factor-β; TIMPs, tissue inhibitor of metalloproteinases; MMPs, matrix metalloproteinases; TRAIL, TNF-related apoptosis-inducing ligand.

the injury [152]. Although HSCs are classically considered to be a major source of myofibroblasts [154, 155], other cell types like portal myofibroblasts and cells recruited from the bone marrow also contribute to the expansion of the myofibroblast population observed during the liver injury [154]. Activated HSCs also secrete an increased amount of MMPs and their inhibitors, TIMPs, which are necessary for the ECM remodeling [154, 156]. HSC activation leads to the up-regulation of TIMPs and TGF-β1 with the inhibition of MMP activity. The TIMP activation thus stimulates collagen type I synthesis and ECM deposition in the extracellular space [157]. Besides injured hepatocytes, hepatic macrophages (KCs), endothelial cells, and lymphocytes

**Figure 1.** Imbalances in ECM synthesis and degradation result in liver fibrosis. Regulation of degradation is determined by the balance between the activity of MMPs and TIMPs. The MMPs degrade both collagenous and non-collagenous components of ECM in the extracellular space. MMP activity is regulated by TIMPs, which bind to MMPs, blocking their proteolytic activity. Increase in the TIMPs/MMPs ratio in liver promotes fibrosis by protecting accumulated matrix from degradation by MMPs. ECM, extracellular matrix; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitor of

HSC activation is still the primary pathway leading to the liver fibrosis and it consists of two main stages: initiation and perpetuation (**Figure 2**) [126]. The initiation stage is related with the early changes in gene expression and phenotype that render the cells responsive to several cytokines and stimuli. Initiation of HSC activation is stimulated by several soluble factors such as oxidant stress signals (ROS), apoptotic bodies, and paracrine stimuli from neighboring cell types including hepatocytes, KCs, sinusoidal endothelium, and platelets [8, 72]. Hepatocytes

also drive HSC activation [158].

268 Liver Cirrhosis - Update and Current Challenges

metalloproteinases.

are believed to represent a major source of ROS as well as of other oxidative stress-related reactive mediators or intermediates [1]. Hepatocyte apoptosis leads to the release of cellular contents such as DNA and ROS that activate KCs to release pro-inflammatory (such as TNF-α, IL-1β, IL-6, MCP-1) and pro-fibrogenic (especially TGF-β) factors [158]. Hepatocyte apoptosis following injury also promotes initiation of HSC activation through a process mediated by Fas, and this process may involve the TRAIL [159]. After stimulation by cytokines or engulfment of apoptotic bodies, KCs stimulate matrix synthesis and cell proliferation through the actions of cytokines including TGF-β1 and ROS/lipid peroxides [64]. Endothelial cells are also likely to participate by conversion of TGF-β from the latent to the active, pro-fibrogenic form [126]. Platelets are another important source of paracrine stimuli, including PDGF, TGF-β1, and EGF [126]. On the other hand, perpetuation stage results from the effects of these stimuli on maintaining the activated phenotype and generating liver fibrosis. This stage involves autocrine as well as paracrine cycles. It includes HSC activation and related cellular changes such as proliferation, chemotaxis, fibrogenesis, contractility, and matrix degradation [126]. Activated HSCs proliferate in response to various kinds of cytokines, chemokines, and growth factors such as TGF-β, EGF, and PDGF [2, 8]. TGF-β, which has been identified as the most pro-fibrotic cytokine, promotes expression of collagen type I by activated HSCs and inhibits ECM degradation through the expression of TIMPs [160]. In parallel, PDGF has emerged as the most potent proliferative cytokine for HSCs [8]. Also, activated HSCs show chemotactic response, migrate toward damaged area and start to accumulate [3]. They express the cytoskeleton protein (α-SMA), equipping the cells with a contractile apparatus and collagens (especially type I) [12, 161, 162]. Thus, HSCs are able to constrict individual sinusoids as well as the entire fibrotic liver [3]. The net effect of these changes is to increase ECM deposition. In addition, cytokine release by HSCs can expand the inflammatory and fibrogenic tissue responses, and matrix proteases may hasten the replacement of normal matrix with fibrotic scar [126]. Briefly, activated HSCs are major effectors of liver fibrogenesis by integrating all incoming paracrine or autocrine signals released from both parenchymal and non-parenchymal cells (pro-inflammatory cytokines, growth factors, chemokines, ROS, and others).

Chronic inflammation and fibrosis are inseparably linked and the interactions between immune cells, local fibroblasts and especially subsets of macrophages determine the outcome of liver injury [8]. Macrophage phenotype and function are critical determinants of fibrotic scarring or resolution of injury. Macrophages, which are typically categorized into classically activated (M1) or alternatively activated (M2) phenotypes, play dual roles in the progression and resolution of liver fibrosis [163]. Typically, M1 macrophages play a pro-inflammatory role in liver injury and produce inflammatory cytokines, while M2 macrophages exert an anti-inflammatory role during tissue repair and fibrosis. The imbalance of M1 and M2 macrophages mediates the progression and resolution of liver fibrosis [164]. During the early stages of liver injury, bone marrow-derived monocytes are extensively recruited to the liver and then differentiate into inflammatory macrophages (mostly M1 macrophages) to produce pro-inflammatory and profibrotic cytokines, thereby promoting inflammatory responses and HSC activation. Afterwards, recruited macrophages switch their phenotypes (mostly M2 macrophages) to secrete MMPs for the successful resolution in hepatic scar [153, 165, 166]. Therefore, a complicated interplay between M1 and M2 types of macrophages plays a critical role in fibrogenesis [128].

#### **6.3. Liver fibrosis is potentially reversible**

Liver fibrosis is thought to be a potentially reversible condition if the cause of liver injury is removed (such as virus suppression or alcohol absence) (**Figure 2**). Regression of liver fibrosis is associated either with elimination of activated HSCs via apoptosis or senescence or with reversion of activated HSCs to a more quiescent phenotype. It has been shown that HSCs are sensitive to Fas and TRAIL-mediated apoptosis, and natural killer cells can induce apoptosis of HSCs by a TRAIL-mediated mechanism [167]. Similarly, TRAIL expressed by KCs is also thought to mediate HSC apoptosis [168]. In addition, apoptosis of activated HSCs is for sure followed by a decrease in collagen production as well as a reduction in TIMP synthesis with an increase in the hepatic MMP expression [1]. Therefore, activated HSCs, the primary source of ECM, are the most attractable target for reversing liver fibrosis [169].

## **7. Halofuginone**

autocrine as well as paracrine cycles. It includes HSC activation and related cellular changes such as proliferation, chemotaxis, fibrogenesis, contractility, and matrix degradation [126]. Activated HSCs proliferate in response to various kinds of cytokines, chemokines, and growth factors such as TGF-β, EGF, and PDGF [2, 8]. TGF-β, which has been identified as the most pro-fibrotic cytokine, promotes expression of collagen type I by activated HSCs and inhibits ECM degradation through the expression of TIMPs [160]. In parallel, PDGF has emerged as the most potent proliferative cytokine for HSCs [8]. Also, activated HSCs show chemotactic response, migrate toward damaged area and start to accumulate [3]. They express the cytoskeleton protein (α-SMA), equipping the cells with a contractile apparatus and collagens (especially type I) [12, 161, 162]. Thus, HSCs are able to constrict individual sinusoids as well as the entire fibrotic liver [3]. The net effect of these changes is to increase ECM deposition. In addition, cytokine release by HSCs can expand the inflammatory and fibrogenic tissue responses, and matrix proteases may hasten the replacement of normal matrix with fibrotic scar [126]. Briefly, activated HSCs are major effectors of liver fibrogenesis by integrating all incoming paracrine or autocrine signals released from both parenchymal and non-parenchymal cells (pro-inflammatory cytokines, growth factors, chemokines, ROS, and others).

Chronic inflammation and fibrosis are inseparably linked and the interactions between immune cells, local fibroblasts and especially subsets of macrophages determine the outcome of liver injury [8]. Macrophage phenotype and function are critical determinants of fibrotic scarring or resolution of injury. Macrophages, which are typically categorized into classically activated (M1) or alternatively activated (M2) phenotypes, play dual roles in the progression and resolution of liver fibrosis [163]. Typically, M1 macrophages play a pro-inflammatory role in liver injury and produce inflammatory cytokines, while M2 macrophages exert an anti-inflammatory role during tissue repair and fibrosis. The imbalance of M1 and M2 macrophages mediates the progression and resolution of liver fibrosis [164]. During the early stages of liver injury, bone marrow-derived monocytes are extensively recruited to the liver and then differentiate into inflammatory macrophages (mostly M1 macrophages) to produce pro-inflammatory and profibrotic cytokines, thereby promoting inflammatory responses and HSC activation. Afterwards, recruited macrophages switch their phenotypes (mostly M2 macrophages) to secrete MMPs for the successful resolution in hepatic scar [153, 165, 166]. Therefore, a complicated interplay

between M1 and M2 types of macrophages plays a critical role in fibrogenesis [128].

of ECM, are the most attractable target for reversing liver fibrosis [169].

Liver fibrosis is thought to be a potentially reversible condition if the cause of liver injury is removed (such as virus suppression or alcohol absence) (**Figure 2**). Regression of liver fibrosis is associated either with elimination of activated HSCs via apoptosis or senescence or with reversion of activated HSCs to a more quiescent phenotype. It has been shown that HSCs are sensitive to Fas and TRAIL-mediated apoptosis, and natural killer cells can induce apoptosis of HSCs by a TRAIL-mediated mechanism [167]. Similarly, TRAIL expressed by KCs is also thought to mediate HSC apoptosis [168]. In addition, apoptosis of activated HSCs is for sure followed by a decrease in collagen production as well as a reduction in TIMP synthesis with an increase in the hepatic MMP expression [1]. Therefore, activated HSCs, the primary source

**6.3. Liver fibrosis is potentially reversible**

270 Liver Cirrhosis - Update and Current Challenges

Halofuginone, a non-toxic and low molecular weight plant alkaloid [7-bromo-6-chloro-3-(3 hydroxy-2-piperidine)-2-oxopropyl-4(3H)-quinazoline] (**Figure 3**) isolated from the roots of *Dichroa febrifuga* (Chinese medicinal plant), is used worldwide as an anti-parasitic drug in commercial poultry production [16]. At first, halofuginone was identified as a potent inhibitor of collagen type α1 gene expression and ECM deposition. At present, it is being evaluated in clinical trial for Duchenne muscular dystrophy, in which fibrosis is the main complication.

### **7.1. Halofuginone and its effect on collagen synthesis**

Halofuginone was found to be a potent inhibitor of collagen type α1 gene expression [17], which was demonstrated in a broad range of cell types including rat, mouse, chicken, and human, both *in vitro* and *in vivo* [16–20]. The discovery of the inhibitory effect of halofuginone on collagen synthesis and ECM deposition has led to intensive studies that were aimed to control many diseases associated with excessive collagen accumulation, such as pulmonary, pancreatic and renal fibrosis [21–23], scleroderma and chronic graft-versus-host disease [24], post-operative peritendinous and abdominal adhesions [25, 26], urethral and esophageal strictures [27, 28], wound repair [29], burn injury [30], renal injury [31, 32], injury-induced arterial intimal hyperplasia [33], colitis [34], and liver fibrosis and cirrhosis [35–39]. Inhibition is independent of the route of administration (intraperitoneally, administered locally, or given orally).

Halofuginone was found to inhibit collagen type I synthesis but not that of type II [17] or III [170] *in vitro*. The inhibitor effect of halofuginone on collagen α1 synthesis appears not to be a direct effect but rather dependent on new protein synthesis, because concurrent treatment of fibroblasts with protein synthesis inhibitors blocks the suppressive effect of halofuginone on collagen α1 mRNA gene expression [18].

Because of the significant impact of fibrosis on human health, there is an unmet need for safe and effective therapies that directly target fibrosis. In animal models of fibrosis, regardless of the tissue, halofuginone had a minimal effect on collagen levels in the control (non-fibrotic) animals; however, it displayed a strong inhibitory effect in the fibrotic organs. This suggests that the regulation of the low-level expression of collagen type I genes differs from that of the

**Figure 3.** Chemical structure of halofuginone.

overexpression induced by the fibrogenic stimulus, which is usually an aggressive and rapid process [171]. Halofuginone mainly affects the stimulated collagen synthesis, therefore, when it is administered systemically, it is actually targeted to the desired fibrotic location without affecting collagen synthesis in other regions.

#### **7.2. Halofuginone and TGF-β pathway**

TGF-β is a "master switch" in chronic liver disease, being involved in all stages of the disease progression, from initial liver injury, inflammation, fibrosis, to cirrhosis and hepatocellular carcinoma at the end [172]. TGF-β signals through transmembrane receptor serine/threonine kinases to activate novel signaling intermediates called Smad proteins, which then modulate transcription of target genes [173]. TGF-β, signaling via Smad3, is the most pro-fibrogenic cytokine present in the liver and the major promoter of ECM synthesis [173, 174]. It induces pro-fibrotic cellular and transcriptional responses such as induction of the synthesis of ECM components, especially collagen, as well as fibronectin and laminin, and it inhibits the matrix degradation enzymes [175]. In various experimental fibrotic models, no effect of halofuginone was observed on the expression of the TGF-β receptors gene or on TGF-β levels [176–178]. This finding supports the hypothesis that the halofuginone target is down-stream in the TGF-β pathway. Halofuginone is an inhibitor of Smad3 phosphorylation down-stream of the TGF-β signaling pathway [177, 179, 180]. In chemically induced liver fibrosis, halofuginone affects TGF-β regulated genes through inhibition of Smad3 phosphorylation of activated HSCs [181]. It inhibits TGF-β-induced phosphorylation of Smad3 and also increases the expression of the inhibitory Smad7 in several cell types (such as fibroblasts, hepatic and pancreatic stellate cells, tumor cells and myoblasts) [178, 181–183]. The inhibition of Smad3 phosphorylation is associated with the halofuginone-dependent activation of Akt MAPK/ERK and p38 MAPK phosphorylation [182]. Thus, drugs that selectively target individual signaling pathways down-stream of the TGF-β receptor are likely to be more successful.

#### **7.3. Halofuginone affects pre-existing fibrosis**

Halofuginone affects fibrosis as a preventive agent when it was administered before or together with the fibrotic stimulus [21, 26, 27, 35, 184]. It can elicit resolution of established fibrosis, a capability that sets it apart from all other preventive anti-fibrotic agents. For example, in rats with established thioacetamide-induced liver fibrosis, addition of halofuginone to the diet results in almost complete resolution of the fibrotic condition as measured by hydroxyproline levels in the liver [36]. This is probably due to up-regulation of the collagen degradation pathway by inhibition of the TIMP-1, and activation of MMPs [43]. In addition, halofuginone given orally before fibrosis induction prevents the activation of most of the stellate cells and the remaining cells expressed low levels of collagen α1 gene, resulting in low levels of collagen [36]. Furthermore, halofuginone administration in low concentrations prior to and following partial hepatectomy in cirrhotic rats does not inhibit normal liver regeneration, despite the reduced levels of collagen type I mRNA [37]. When given to rats with established fibrosis, halofuginone causes significant reductions in α-SMA, TIMP-2, collagen type I gene expression, and collagen accumulation [37]. These animals demonstrate improved capacity for regeneration, suggesting the possible beneficial use of halofuginone before and during fibrotic/cirrhotic liver regeneration.

#### **7.4. Halofuginone as an anti-fibrotic agent**

overexpression induced by the fibrogenic stimulus, which is usually an aggressive and rapid process [171]. Halofuginone mainly affects the stimulated collagen synthesis, therefore, when it is administered systemically, it is actually targeted to the desired fibrotic location without

TGF-β is a "master switch" in chronic liver disease, being involved in all stages of the disease progression, from initial liver injury, inflammation, fibrosis, to cirrhosis and hepatocellular carcinoma at the end [172]. TGF-β signals through transmembrane receptor serine/threonine kinases to activate novel signaling intermediates called Smad proteins, which then modulate transcription of target genes [173]. TGF-β, signaling via Smad3, is the most pro-fibrogenic cytokine present in the liver and the major promoter of ECM synthesis [173, 174]. It induces pro-fibrotic cellular and transcriptional responses such as induction of the synthesis of ECM components, especially collagen, as well as fibronectin and laminin, and it inhibits the matrix degradation enzymes [175]. In various experimental fibrotic models, no effect of halofuginone was observed on the expression of the TGF-β receptors gene or on TGF-β levels [176–178]. This finding supports the hypothesis that the halofuginone target is down-stream in the TGF-β pathway. Halofuginone is an inhibitor of Smad3 phosphorylation down-stream of the TGF-β signaling pathway [177, 179, 180]. In chemically induced liver fibrosis, halofuginone affects TGF-β regulated genes through inhibition of Smad3 phosphorylation of activated HSCs [181]. It inhibits TGF-β-induced phosphorylation of Smad3 and also increases the expression of the inhibitory Smad7 in several cell types (such as fibroblasts, hepatic and pancreatic stellate cells, tumor cells and myoblasts) [178, 181–183]. The inhibition of Smad3 phosphorylation is associated with the halofuginone-dependent activation of Akt MAPK/ERK and p38 MAPK phosphorylation [182]. Thus, drugs that selectively target individual signaling pathways

Halofuginone affects fibrosis as a preventive agent when it was administered before or together with the fibrotic stimulus [21, 26, 27, 35, 184]. It can elicit resolution of established fibrosis, a capability that sets it apart from all other preventive anti-fibrotic agents. For example, in rats with established thioacetamide-induced liver fibrosis, addition of halofuginone to the diet results in almost complete resolution of the fibrotic condition as measured by hydroxyproline levels in the liver [36]. This is probably due to up-regulation of the collagen degradation pathway by inhibition of the TIMP-1, and activation of MMPs [43]. In addition, halofuginone given orally before fibrosis induction prevents the activation of most of the stellate cells and the remaining cells expressed low levels of collagen α1 gene, resulting in low levels of collagen [36]. Furthermore, halofuginone administration in low concentrations prior to and following partial hepatectomy in cirrhotic rats does not inhibit normal liver regeneration, despite the reduced levels of collagen type I mRNA [37]. When given to rats with established fibrosis, halofuginone causes significant reductions in α-SMA, TIMP-2, collagen type I gene expression, and collagen accumulation [37]. These animals demonstrate improved capacity for regeneration, suggesting the possible benefi-

down-stream of the TGF-β receptor are likely to be more successful.

cial use of halofuginone before and during fibrotic/cirrhotic liver regeneration.

**7.3. Halofuginone affects pre-existing fibrosis**

affecting collagen synthesis in other regions.

**7.2. Halofuginone and TGF-β pathway**

272 Liver Cirrhosis - Update and Current Challenges

In recent years, much attention was focused on halofuginone against liver fibrosis (**Table 3**). Although the exact anti-fibrotic mechanism of halofuginone is not well understood, it is found to be associated with inhibition of TGF-β signaling [179], which is known to inhibit mesengial


*Abbreviations*: DMN, dimethylnitrosamine; TAA, thioacetamide; TIMP, tissue inhibitor of metalloproteinase; SMA, smooth muscle actin; ConA, Concanavalin A; Th17, T helper 17; TGF-β, transforming growth factor-β; MMP, matrix metalloproteinase; HSC, hepatic stellate cell; p38 MAPK, p38 mitogen-activated protein kinase; NF-κB, nuclear factorκB; IGFBP-1, insulin-like growth factor binding protein-1; PRL-1, tyrosine phosphatase; IFN-γ, interferon-γ; IL-2, interleukin-2; HCC, hepatocellular carcinoma.

**Table 3.** Effects of halofuginone in various experimental liver diseases.

cell proliferation and ECM deposition [185]. In several animal models of fibrosis, in which excess collagen is the characteristic of the disease, halofuginone prevents transition of the fibroblasts to myofibroblasts by inhibition of Smad3 phosphorylation down-stream of the TGF-β signaling pathway [186, 187], thereby inhibits collagen synthesis [186]. Halofuginone also regulates cell growth and differentiation, apoptosis, cell migration, and immune cell function [41]. It prevents concanavalin A-induced liver fibrosis by affecting Th17 cell differentiation, which suggests a direct link between the myofibroblasts/fibrosis pathway and the Th17 pro-inflammatory pathway [38]. Th17 cells, a distinct subset of CD4<sup>+</sup> T cells with IL-17 as their major cytokine, orchestrate the pathogenesis of inflammation [171]. It has been suggested that halofuginone-dependent inhibition of fibrosis includes selective inhibition of the Th17 cell development by activating the amino acid starvation response [188, 189]. Halofuginone activates the amino acid starvation response by directly inhibiting the prolyl-tRNA synthetase activity of glutamyl-prolyl-tRNA synthetase [190]. Furthermore, addition of exogenous proline reverses a broad range of halofuginone-induced cellular effects, indicating that glutamylprolyl-tRNA synthetase-inhibition underlies the therapeutic activities of halofuginone [190]. TGF-β is required for facilitation of differentiation of the inflammatory Th17 cell subset [191], which suggests the presence of a connection between the TGF-β signaling inhibition and the amino acid starvation response [187]. Treatment with halofuginone also effectively inhibits the delayed-type hypersensitivity response, indicating suppression of T cell–mediated inflammation *in vivo* [42]. Moreover, it was shown that halofuginone is a potent inhibitor of NF-κB, pro-inflammatory cytokines, and p38 MAPK phosphorylation in activated T cells *in vitro* [42]. Also, submicromolar concentrations of halofuginone inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NF-κB. The remarkable induction of MMP-3 and -13 makes halofuginone a promising agent for anti-fibrotic combination therapies [43]. Halofuginone also affects the cross-talk between the hepatocytes and the HSCs by up-regulating the synthesis and secretion of insulin-like growth factor binding protein-1 (IGFBP-1), which inhibits HSC migration [192]. It also affects the expression of early genes of liver regeneration, IGFBP-1 whose synthesis and secretion is regulated in part by TGF-β [192] and tyrosine phosphatase (PRL-1) whose synthesis is regulated by transcription factor early growth response-1 (Egr-1) probably via TGF-β [193].

#### **7.5. Anti-tumoral role of halofuginone**

In many types of tumor, there is a strong relationship between tissue fibrosis and increased risk of tumor development. For example, the leading risk factor for hepatocellular carcinoma is liver cirrhosis, and its associated inflammation, regeneration, and fibrosis [194, 195]. Tumor cells develop and metastasize more effectively in fibrotic tissues; therefore, any reduction in tissue fibrosis reduces the risk of cancer [171]. Halofuginone reduces tumor growth and mortality in xenograph mice implanted with human hepatoma cells [196]. In diethylnitrosamine and *N*-nitrosomorpholine-induced, spontaneously metastasizing hepatocellular carcinoma, halofuginone suppresses lung metastasis in rats through MMP inhibition [197]. Moreover, halofuginone treatment results in effective inhibitory effects on the cascade of events leading to angiogenesis (formation of new blood vessels), such as abrogation of endothelial cell MMP-2 expression, basement membrane invasion, capillary tube formation, vascular sprouting, and deposition of sub-endothelial ECM *in vitro* [171]. Inhibition of angiogenesis is mostly accompanied by inhibition of the fibroblasts to myofibroblasts transition, reduction in tumor stroma ECM, and inhibition of tumor growth [171]. The high effectiveness of halofuginone in reducing fibrosis, which affects tumor growth and tissue regeneration in the liver, arises from its dual role in inhibiting the TGF-β signaling and Th17 cell development [187].

## **8. Conclusion**

cell proliferation and ECM deposition [185]. In several animal models of fibrosis, in which excess collagen is the characteristic of the disease, halofuginone prevents transition of the fibroblasts to myofibroblasts by inhibition of Smad3 phosphorylation down-stream of the TGF-β signaling pathway [186, 187], thereby inhibits collagen synthesis [186]. Halofuginone also regulates cell growth and differentiation, apoptosis, cell migration, and immune cell function [41]. It prevents concanavalin A-induced liver fibrosis by affecting Th17 cell differentiation, which suggests a direct link between the myofibroblasts/fibrosis pathway and the Th17

major cytokine, orchestrate the pathogenesis of inflammation [171]. It has been suggested that halofuginone-dependent inhibition of fibrosis includes selective inhibition of the Th17 cell development by activating the amino acid starvation response [188, 189]. Halofuginone activates the amino acid starvation response by directly inhibiting the prolyl-tRNA synthetase activity of glutamyl-prolyl-tRNA synthetase [190]. Furthermore, addition of exogenous proline reverses a broad range of halofuginone-induced cellular effects, indicating that glutamylprolyl-tRNA synthetase-inhibition underlies the therapeutic activities of halofuginone [190]. TGF-β is required for facilitation of differentiation of the inflammatory Th17 cell subset [191], which suggests the presence of a connection between the TGF-β signaling inhibition and the amino acid starvation response [187]. Treatment with halofuginone also effectively inhibits the delayed-type hypersensitivity response, indicating suppression of T cell–mediated inflammation *in vivo* [42]. Moreover, it was shown that halofuginone is a potent inhibitor of NF-κB, pro-inflammatory cytokines, and p38 MAPK phosphorylation in activated T cells *in vitro* [42]. Also, submicromolar concentrations of halofuginone inhibit HSC proliferation and migration and up-regulate their expression of fibrolytic MMP-3 and -13 via activation of p38 MAPK and NF-κB. The remarkable induction of MMP-3 and -13 makes halofuginone a promising agent for anti-fibrotic combination therapies [43]. Halofuginone also affects the cross-talk between the hepatocytes and the HSCs by up-regulating the synthesis and secretion of insulin-like growth factor binding protein-1 (IGFBP-1), which inhibits HSC migration [192]. It also affects the expression of early genes of liver regeneration, IGFBP-1 whose synthesis and secretion is regulated in part by TGF-β [192] and tyrosine phosphatase (PRL-1) whose synthesis is regulated by transcription factor early growth response-1 (Egr-1) probably via TGF-β [193].

In many types of tumor, there is a strong relationship between tissue fibrosis and increased risk of tumor development. For example, the leading risk factor for hepatocellular carcinoma is liver cirrhosis, and its associated inflammation, regeneration, and fibrosis [194, 195]. Tumor cells develop and metastasize more effectively in fibrotic tissues; therefore, any reduction in tissue fibrosis reduces the risk of cancer [171]. Halofuginone reduces tumor growth and mortality in xenograph mice implanted with human hepatoma cells [196]. In diethylnitrosamine and *N*-nitrosomorpholine-induced, spontaneously metastasizing hepatocellular carcinoma, halofuginone suppresses lung metastasis in rats through MMP inhibition [197]. Moreover, halofuginone treatment results in effective inhibitory effects on the cascade of events leading to angiogenesis (formation of new blood vessels), such as abrogation of endothelial cell MMP-2 expression, basement membrane invasion, capillary tube formation, vascular sprouting, and

T cells with IL-17 as their

pro-inflammatory pathway [38]. Th17 cells, a distinct subset of CD4<sup>+</sup>

274 Liver Cirrhosis - Update and Current Challenges

**7.5. Anti-tumoral role of halofuginone**

Fibrosis is a pathological process associated with excessive ECM deposition that leads to destruction of organ architecture and function. Fibrosis contributes enormously to deaths worldwide; thus, effective therapies are of a great need. Halofuginone has great potential as an anti-fibrotic therapeutic. Systemic administration of halofuginone in animal models and humans is well tolerated [24]. Additionally, in most animal models of fibrosis, halofuginone has a minimal effect on collagen levels in non-fibrotic animals, while exerting strong inhibitory effects in fibrotic organs. It mainly affects stimulated collagen synthesis without altering the usual low physiological level of collagen expression. Because halofuginone inhibits collagen type I synthesis on the transcriptional level and reduces ECM deposition, it is a promising candidate for treatment of diseases associated with excessive ECM, such as liver fibrosis/ cirrhosis. Thus, halofuginone meets the criteria as a promising anti-fibrotic drug for further evaluation in the treatment of liver fibrosis/cirrhosis.

#### **Conflicts of Interest**

The author reports no conflicts of interest.

## **Author details**

#### Berna Karakoyun

Address all correspondence to: bernakarakoyun@gmail.com; berna.lacin@marmara.edu.tr

Department of Basic Health Sciences, Faculty of Health Sciences, Marmara University, Istanbul, Turkey

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10.1371/journal.pone.0022770


## *Edited by Georgios Tsoulfas*

Liver cirrhosis represents one of the major challenges for most physicians and surgeons on a global scale. This book provides practicing hepatologists, gastroenterologists and liver surgeons with a valuable tool in their efforts to understand the (molecular) mechanisms involved, be updated regarding the newest and less invasive diagnostic methods, and educate themselves about the challenges involved in the management of liver cirrhosis and its complications. The authors of this book represent a team of true global experts on the topic. In addition to the knowledge shared, the authors provide their personal clinical experience on a variety of different aspects of liver cirrhosis, giving us a well-rounded overview.

Liver Cirrhosis - Update and Current Challenges

Liver Cirrhosis

Update and Current Challenges

*Edited by Georgios Tsoulfas*

Photo by klickit24 / iStock