**3. Is cellular senescence important in the development of hepatic diseases?**

Aging changes the biological processes in many organs and tissues of mammalians, leading to the development of age-associated diseases and aberrant body homeostasis. Nearly 13% of the population in the United States is over the age of 65 years; this percentage and the number of elderly people will increase substantially over the next 50 years [84]. Although the aging process does not cause death, elderly people appear to be predisposed to a variety of diseases; therefore, aging facilitates the onset and/or progression of various pathologies including diseases of the liver [84]. The ability to withstand hepatic insult decreases each decade, including those associated with high triglyceride levels, nonalcoholic fatty liver diseases, and HCC [85–87]. The percentage of deaths caused by liver diseases increases dramatically in humans over the age of 45 years. Accordingly, age is a critical determinant in the outcome of hepatitis C infection [88] and liver transplantation [89]. However, the most dramatic effect of aging on the liver is its delayed and/or reduced proliferation after a loss of mass from a surgical or chemical injury [90–92]. Based on these correlations, it has been suggested that elderly people are predisposed to liver diseases. The livers of young individuals regenerate after these treatments and maintain homeostasis; conversely, there is a dramatically reduced regeneration of the livers of older people. The clinical outcome of human acute liver injury is also partially dependent on the potential for hepatic regeneration. In older people, there is a higher incidence of acute liver failure and a higher mortality with acute hepatitis A [93, 94]. In a cohort of patients with acute liver failure secondary to viral hepatitis, age was an independent predictor of a poor outcome, with the worst outcomes in people over the age of 50 years [95]. Wong et al. and Poynard et al. were among the first to demonstrate that age at infection was a major risk factor for subsequent fibrosis progression and that the rate of fibrosis progression accelerated with increasing age [96, 97]. Studies of other liver diseases have demonstrated similar findings. Age is an independent risk factor for poor outcomes in primary biliary cirrhosis (PBC) in addition to the presence of portal hypertension and impaired liver function [98]. Similarly, age, independent of bilirubin, prothrombin time, and renal function, predicted the outcome in patients with alcoholic hepatitis [99]. There is some evidence that age may influence disease progression in nonalcoholic fatty liver disease (NAFLD) [100, 101].

The role of CS in diverse chronic liver diseases has come into recent research. The structure of hepatocytes changes with age. The volume of hepatocytes increases with development and maturation but declines with senescence [84]. The relative volumes of hepatocyte organelles also change during aging. The universal change in diagnostic liver biopsy specimens is the age-related cytoplasmic accumulation of highly oxidized insoluble protein lipofuscin or age pigment, which in turn reflects a concomitant increase in the volume of the dense body compartment [102]. Accelerated hepatocyte aging and the accumulation of senescent hepatocytes have been found in diverse chronic liver disorders [103–108]. Furthermore, the studies have shown that the increased proportion of senescent hepatocytes is associated independently with increased fibrosis stage, impaired hepatic function, and an adverse liver-related outcome including liver-related death [103–109]. However, it is still unknown whether hepatocyte senescence contributes causally to fibrosis progression, declining hepatic function, or the increased risk of an adverse liver-related outcome.

#### **3.1. Hepatic fibrosis**

senescence/ROS is a transitory, sporadic, or specific event of a determined HCC development stage. We have recently shown that, concomitant to the HCC development induced by diethylnitrosamine, there is also a chronological appearance of CS [69]; however, its association with ROS generation and effect on the outcome of multifocal HCC type has not been investigated.

Aging changes the biological processes in many organs and tissues of mammalians, leading to the development of age-associated diseases and aberrant body homeostasis. Nearly 13% of the population in the United States is over the age of 65 years; this percentage and the number of elderly people will increase substantially over the next 50 years [84]. Although the aging process does not cause death, elderly people appear to be predisposed to a variety of diseases; therefore, aging facilitates the onset and/or progression of various pathologies including diseases of the liver [84]. The ability to withstand hepatic insult decreases each decade, including those associated with high triglyceride levels, nonalcoholic fatty liver diseases, and HCC [85–87]. The percentage of deaths caused by liver diseases increases dramatically in humans over the age of 45 years. Accordingly, age is a critical determinant in the outcome of hepatitis C infection [88] and liver transplantation [89]. However, the most dramatic effect of aging on the liver is its delayed and/or reduced proliferation after a loss of mass from a surgical or chemical injury [90–92]. Based on these correlations, it has been suggested that elderly people are predisposed to liver diseases. The livers of young individuals regenerate after these treatments and maintain homeostasis; conversely, there is a dramatically reduced regeneration of the livers of older people. The clinical outcome of human acute liver injury is also partially dependent on the potential for hepatic regeneration. In older people, there is a higher incidence of acute liver failure and a higher mortality with acute hepatitis A [93, 94]. In a cohort of patients with acute liver failure secondary to viral hepatitis, age was an independent predictor of a poor outcome, with the worst outcomes in people over the age of 50 years [95]. Wong et al. and Poynard et al. were among the first to demonstrate that age at infection was a major risk factor for subsequent fibrosis progression and that the rate of fibrosis progression accelerated with increasing age [96, 97]. Studies of other liver diseases have demonstrated similar findings. Age is an independent risk factor for poor outcomes in primary biliary cirrhosis (PBC) in addition to the presence of portal hypertension and impaired liver function [98]. Similarly, age, independent of bilirubin, prothrombin time, and renal function, predicted the outcome in patients with alcoholic hepatitis [99]. There is some evidence that age may influence disease progression in nonalcoholic fatty

The role of CS in diverse chronic liver diseases has come into recent research. The structure of hepatocytes changes with age. The volume of hepatocytes increases with development and maturation but declines with senescence [84]. The relative volumes of hepatocyte organelles also change during aging. The universal change in diagnostic liver biopsy specimens is the

This is an interesting and comprehensive proposal worth investigating.

**3. Is cellular senescence important in the development of** 

**hepatic diseases?**

126 Senescence - Physiology or Pathology

liver disease (NAFLD) [100, 101].

Hepatic fibrosis is a common pathological consequence of chronic liver diseases and results from the progressive accumulation of an excess quantity of extracellular matrix (ECM) tissue. In many patients, continuous liver parenchyma insult ultimately leads to cirrhosis [110]. The ECM accumulation during chronic liver injury is caused by a heterogeneous population of myofibroblasts derived mainly from hepatic stellate cells and from resident fibroblasts. Myofibroblasts are highly proliferative cells with enhanced survival that migrate and accumulate at sites of liver damage in response to a liver insult that caused a wide variety of growth factors, cytokines, and lipid mediators. Recently, many studies have observed that the presence of an increased proportion of senescent hepatocytes is associated with an increased fibrosis stage. This results in impaired hepatic function and an adverse liver-related outcome, including liver-related death [103, 104, 106–108].

#### **3.2. Cirrhosis and HCC**

In humans, cirrhosis is induced by a variety of liver diseases. Regardless of its etiology, cirrhosis evolves slowly over many years, and chronic hepatocyte death and renewal are major predisposing factors [111]. Cirrhosis is associated with life-threatening complications due to a decreased functional parenchymal reserve and altered hepatic blood flow. A recent report by WHO indicates that cirrhosis accounts for 170,000 deaths in Europe per year [110]. The cellular growth arrest and CS appear to be in a pro-fibrotic state. There is existing research on the relation between cirrhosis and telomere shortening, showing that telomere shortening is a marker of cirrhosis and correlates with senescence-associated β-galactosidase in 84% of cirrhosis samples, specifically in hepatocytes [110]. CS contributes to age-related tissue dysfunction, macroH2A1, and a variant of histone H2A. CS is also a marker of senescence-associated heterochromatic foci that synergizes with DNA methylation to silence tumor-suppressor genes in human fibroblasts. MacroH2A1, a variant of histone H2A, is a marker of senescence-associated heterochromatic foci that synergizes with DNA methylation to silence tumor-suppressor genes in human fibroblasts. In a recent study, Borghesan et al. investigated the relationship between macroH2A1 splice variants, macroH2A1.1 and macroH2A1.2, and liver carcinogenesis. It was found that protein levels of both macroH2A1 isoforms were increased in the livers of very elderly rodents and humans, and were strong immunohistochemical markers of human cirrhosis and HCC [112].

### **3.3. Chronic hepatitis B**

Chronic hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis, and HCC. HBV infection is associated with age, cell cycle arrest, and CS. In a recent study, the authors assessed the HBV antigen production in relation to cell cycle arrest and CS *in vitro* using hepG2 and hepG2.2.15 cell lines [113].The authors found that cell cycle arrest induced *in vitro* by the addition of H<sup>2</sup> O2 caused increased levels of supernatant HBsAg and HBV DNA and increased expression of HBcAg. In contrast, there was no observed effect on HBsAg or HBV DNA production in senescent cells, with only a minor increase in cytoplasmic HBcAg staining [113].Widespread telomere shortening is consistent with accelerated aging in chronic HBV [114].

### **3.4. Chronic hepatitis C**

Chronic infection with hepatitis C virus (HCV) affects approximately 170 million people around the world. Moreover, 20% of these affected people develop cirrhosis and are at great risk to develop HCC. An increased risk of HCV-related cirrhosis is associated with hepatic steatosis, older age, and higher alcohol consumption [88, 115]. It has been reported that during chronic HCV infection, telomere shortening is present in liver tissue [116], likely due to increased hepatocyte turnover. Critically short telomeres trigger replicative senescence. Marshall et al. found a strong correlation between hepatocyte G1 arrest, dysfunctional hepatic regeneration, and increased cirrhosis in patients with chronic HCV. They showed increased hepatocyte cell cycle arrest measured by the expression of the mini-chromosome maintenance protein 2 (Mcm-2) in liver biopsy samples. The researchers also found that p21 expressed predominantly in hepatocytes and it was correlated with the stage of fibrosis [107].

#### **3.5. Nonalcohol-related fatty liver disease (NAFLD)**

NAFLD is a leading cause of chronic liver disease worldwide [117], which presents a wide range of liver disorders including simple steatosis, steatohepatitis, cirrhosis, and HCC [117]. In ob/ob mice, NAFLD is associated with an increased expression of the cell cycle inhibitor p21 and impaired liver regeneration [118]. The combination of impaired regeneration and increased incidence of cancer in NAFLD suggests accelerated aging. In the literature, NAFLD has been separately described by the appearance of premature telomere shortening, increased hepatocyte nuclear area, advanced fibrosis, and p21 expression [103, 104]. Recently, Aravinthan et al. performed a study on 105 archived formalin-fixed paraffin-embedded liver needle biopsy specimens from 70 patients within the spectrum of NAFLD, and 43 liver needle biopsies at the time of liver transplantation from age- and sex-matched donor livers served as controls. In this study, the authors found a close correlation between hepatic steatosis and short telomeres; additionally, the proportion of hepatocytes with DNA damage identified by the presence of γ-H2AX increased in parallel with steatosis grade. The authors suggested that the accumulation of fat in hepatocytes causes DNA damage and telomere erosion, possibly mediated by oxidative stress. A critically short telomere and a break in double-stranded DNA lead to CS and permanent cell cycle arrest. The authors also found a pattern of predominant hepatocyte G1/S phase cell cycle arrest in NAFLD, with an increased expression of Mcm-2 compared to a normal liver. Furthermore, there was increased expression of the p21 protein. A striking feature was the association of p21 expression with the fibrosis stage, which suggests an accumulation of senescent hepatocytes with disease progression [105].

### **3.6. Alcohol-related liver disease**

**3.3. Chronic hepatitis B**

128 Senescence - Physiology or Pathology

chronic HBV [114].

**3.4. Chronic hepatitis C**

induced *in vitro* by the addition of H<sup>2</sup>

Chronic hepatitis B virus (HBV) infection can lead to the development of chronic hepatitis, cirrhosis, and HCC. HBV infection is associated with age, cell cycle arrest, and CS. In a recent study, the authors assessed the HBV antigen production in relation to cell cycle arrest and CS *in vitro* using hepG2 and hepG2.2.15 cell lines [113].The authors found that cell cycle arrest

HBV DNA and increased expression of HBcAg. In contrast, there was no observed effect on HBsAg or HBV DNA production in senescent cells, with only a minor increase in cytoplasmic HBcAg staining [113].Widespread telomere shortening is consistent with accelerated aging in

Chronic infection with hepatitis C virus (HCV) affects approximately 170 million people around the world. Moreover, 20% of these affected people develop cirrhosis and are at great risk to develop HCC. An increased risk of HCV-related cirrhosis is associated with hepatic steatosis, older age, and higher alcohol consumption [88, 115]. It has been reported that during chronic HCV infection, telomere shortening is present in liver tissue [116], likely due to increased hepatocyte turnover. Critically short telomeres trigger replicative senescence. Marshall et al. found a strong correlation between hepatocyte G1 arrest, dysfunctional hepatic regeneration, and increased cirrhosis in patients with chronic HCV. They showed increased hepatocyte cell cycle arrest measured by the expression of the mini-chromosome maintenance protein 2 (Mcm-2) in liver biopsy samples. The researchers also found that p21 expressed pre-

NAFLD is a leading cause of chronic liver disease worldwide [117], which presents a wide range of liver disorders including simple steatosis, steatohepatitis, cirrhosis, and HCC [117]. In ob/ob mice, NAFLD is associated with an increased expression of the cell cycle inhibitor p21 and impaired liver regeneration [118]. The combination of impaired regeneration and increased incidence of cancer in NAFLD suggests accelerated aging. In the literature, NAFLD has been separately described by the appearance of premature telomere shortening, increased hepatocyte nuclear area, advanced fibrosis, and p21 expression [103, 104]. Recently, Aravinthan et al. performed a study on 105 archived formalin-fixed paraffin-embedded liver needle biopsy specimens from 70 patients within the spectrum of NAFLD, and 43 liver needle biopsies at the time of liver transplantation from age- and sex-matched donor livers served as controls. In this study, the authors found a close correlation between hepatic steatosis and short telomeres; additionally, the proportion of hepatocytes with DNA damage identified by the presence of γ-H2AX increased in parallel with steatosis grade. The authors suggested that the accumulation of fat in hepatocytes causes DNA damage and telomere erosion, possibly mediated by oxidative stress. A critically short telomere and a break in double-stranded DNA lead to CS and permanent cell cycle arrest. The authors also found a pattern of predominant hepatocyte G1/S phase cell cycle arrest in NAFLD, with an increased expression of Mcm-2

dominantly in hepatocytes and it was correlated with the stage of fibrosis [107].

**3.5. Nonalcohol-related fatty liver disease (NAFLD)**

O2 caused increased levels of supernatant HBsAg and

Alcohol-related liver disease (ALD) is one of the leading causes of liver-related morbidity and mortality in the world. ALD encompasses a broad spectrum of liver injury from simple steatosis to alcohol-related hepatitis, cirrhosis, and HCC. The pathophysiology of ALD is a complex phenomenon; however, oxidative stress is an important factor in the disease since alcohol consumption increases the production of ROS and diminishes cellular antioxidant levels [119]. The validity of clinical criteria in predicting outcomes in ALD has been studied. In one study, it was shown that the increase of the hepatocyte nuclear area was related to hepatic dysfunction and suggests hepatocyte senescence, since nuclear enlargement is a recognized morphological characteristic of CS [120]. In contrast, irreversible cell arrest that limits the proliferative potential of cell is a hallmark of CS and is mediated by p21 [121]. Thus, p21 has a vital role in the stability of cell cycle arrest and the induction of senescence [121].

#### **3.7. Senolytic drugs**

The accumulation of senescent cells in tissues and organs contributes to age-related diseases; however, the presence of radiations or genotoxic agents may contribute to the development of senescence phenotype. This accumulation is important because senescent cells contribute to alterations in the microenvironment. This can support conditions for diseases such as cancer to develop, but senescent cell quantity is important for organ function in the case of aging.

Senolytic agents may be used for clearance of senescent cells in the organs. Chang et al. used ABT263, a specific inhibitor of apoptosis genes BCL2 and BCL2L1, to selectively induce apoptosis in senescent cells in culture. They carried out experiments using a mouse model with known p16-3 MR and administered ABT263 to mice and observed that p16-positive senescent cells were depleted in bone marrow, lung, and muscle tissue via apoptosis. The data demonstrate that ABT263 is a senolytic drug that acts in a highly specific manner on various tissues to target senescent cells both in culture and *in vivo*. These findings significantly advance clinical targeting of cell senescence [122].
