**3.2. Hepatic stellate cells (HSC) & portal myofibroblasts (MF)**

HSC are quiescent cells that express typical markers of both neural cells and adipocytes (glial fibrillary acid protein-GFAP, peroxisome proliferator-activated receptor gamma-PPAϪ, and adiponectin receptors). They are activated by cytokines, growth factors, ROS, damaged cells and apoptotic bodies [64]. In health, MF are located adjacent to bile duct epithelia and are the first responder to biliary injuries. Upon activation HSC's acquire a MF phenotype, cells that upon phagocytosis of LD and/or apoptotic bodies from damaged cells get additional energy and became Fas-ligand and TNF-α unresponsive to apoptosis; mechanism in use for increase collagen synthesis and deposition [64]. Furthermore, activation of the adenosine receptor A2A increases HSC proliferation and inhibits death and senescence by down regulation of p53 and Rb through the cAMP-PKA/Rac1/p38 MAPK pathway [80]. Activated MF's express CCN1/CYR61, an important regulator of inflammation and wound healing. Cystein-rich 61-protein (CCN1/ CYR61) is a matrix-cellular protein that induces senescence at later stages of wound healing by promoting tissue remodeling through fibrogenic cell apoptosis and attenuation of TGF-ß signaling [81]. HSC and MF senescent fibrogenic cells no longer proliferate, thereby reducing the load of ECM deposition. In addition, senescent fibrogenic cells express an increase in the secretion of metalloproteinases (MTP's) leading to matrix degradation. Apoptotic fragments from HSC and MF are cleared by natural killer cells promoting wound healing, the best characterized mechanism of fibrogenesis resolution [64, 81]. NF-κB is a key regulator for HSC survival and proliferation by maintaining the expression of Mcl-2. Inhibition of NF-κB increases HSC apoptosis by up-regulation of the JNK pathway. Thus, the activation as well as the induction of senescence/ apoptosis of HSC/MF are normal wound healing mechanisms that promote the establishment of normal organ architecture and function with clear paths of initiation and resolution.

During chronic cell injury, such as in a state of high caloric intake enriched with lipids, an increase and progressive pool of biologically active HSC's may become prominent [11]. An incremental chronic state of fibrogenesis alters hepatic architecture leading to a concomitant increase in portal flow resistance, portal hypertension and the development of collateral circulation. In addition, HSC's produce a microenvironment with altered extracellular matrix (ECM) that provides biochemical and mechanical cues to the growth and establishment of tumor cells [67]. Nevertheless, since 90% of the HCC's flourish in a highly progressive fibrotic ECM, the question raises if it is the changes on the microenvironment that further promotes metabolic transformation with an "apoptotic switch" and tumor development. Interestingly, progressive liver fibrogenic ECM becomes enriched with vascular growth factor (VGF) receptor promoting angiogenesis, paving the way for the much needed arterial high O<sup>2</sup> supply for HCC expansion [67].

The different components of the ECM, cellular and non-cellular interact directly and indirectly with malignant cells therefore changing the phenotype of the evolving cells that in turn produces feedback signals to further adapt the microenvironment to the needs of the malignant cell. The link between the actin cytoskeleton and the microenvironment provides an input of intracellular contractile forces capable of regulating signaling pathways fundamental to the definition of cell phenotype, mechanism that constitute the ECM "out-side-in" code to the cell. In response, the anchored cells expressed adhesions molecules and secreted proteins that signals HSC and other ECM regulators increasing anchoring sites in response to the "in-side-out" signaling [67]. Therefore, the metabolic transformation of the already stressed parenchymal cells help to choose a path different to senescence and necrosis but to a path of unregulated regeneration, thus escaping apoptosis. A path that needs an ECM differentiation to assure cell survival in a non-efficient energy redox status.

#### **3.3. Sinusoidal endothelial cells (SEC)**

Cellular events that follows are the activation/repression of factors involved in cell proliferation. In the liver cell, the known transcriptional shift includes activation of FOXO3, FOXII, E2F1, c-jun, C/EBPß, Myb, USF and neutralization of inhibitors of cell proliferation such as Rb family and C/EBP family of proteins [76]. In C/EBP-S193A mice, failure to stop liver regeneration after surgery correlated with the epigenetic repression of C/EBPß, p53, FXR, SIRT1, PGC1α and TERT. The repression was performed by a protein formed by C/EBPß-HDCAC1 complex which also inhibit the promoters of enzymes for glucose synthesis PEPCK and G6P [76]. The response of cell cycle engaged hepatocytes and cell cycle arrested hepatocytes (senescent cell) to injury is different and it may awake an unregulated cell growth on quiescent stem liver cells [76, 77]. Oval shaped liver cells may differentiate into cholangiocytes with a distinct metabolism and perhaps pathway towards malignancy [15, 16]. Although cholangiocytes are metabolically very active cells involved in the secretion and resorption of water and soluble bile components, they are not directly involved in the metabolism and/or regulation of biliary

lipid species (cholesterol, bile acids and phosphatidyl-choline vesicles) [78, 79].

normal organ architecture and function with clear paths of initiation and resolution.

During chronic cell injury, such as in a state of high caloric intake enriched with lipids, an increase and progressive pool of biologically active HSC's may become prominent [11]. An incremental chronic state of fibrogenesis alters hepatic architecture leading to a concomitant increase in portal flow resistance, portal hypertension and the development of collateral circulation. In addition, HSC's produce a microenvironment with altered extracellular matrix (ECM) that provides biochemical and mechanical cues to the growth and establishment of tumor cells [67]. Nevertheless,

HSC are quiescent cells that express typical markers of both neural cells and adipocytes (glial fibrillary acid protein-GFAP, peroxisome proliferator-activated receptor gamma-PPAϪ, and adiponectin receptors). They are activated by cytokines, growth factors, ROS, damaged cells and apoptotic bodies [64]. In health, MF are located adjacent to bile duct epithelia and are the first responder to biliary injuries. Upon activation HSC's acquire a MF phenotype, cells that upon phagocytosis of LD and/or apoptotic bodies from damaged cells get additional energy and became Fas-ligand and TNF-α unresponsive to apoptosis; mechanism in use for increase collagen synthesis and deposition [64]. Furthermore, activation of the adenosine receptor A2A increases HSC proliferation and inhibits death and senescence by down regulation of p53 and Rb through the cAMP-PKA/Rac1/p38 MAPK pathway [80]. Activated MF's express CCN1/CYR61, an important regulator of inflammation and wound healing. Cystein-rich 61-protein (CCN1/ CYR61) is a matrix-cellular protein that induces senescence at later stages of wound healing by promoting tissue remodeling through fibrogenic cell apoptosis and attenuation of TGF-ß signaling [81]. HSC and MF senescent fibrogenic cells no longer proliferate, thereby reducing the load of ECM deposition. In addition, senescent fibrogenic cells express an increase in the secretion of metalloproteinases (MTP's) leading to matrix degradation. Apoptotic fragments from HSC and MF are cleared by natural killer cells promoting wound healing, the best characterized mechanism of fibrogenesis resolution [64, 81]. NF-κB is a key regulator for HSC survival and proliferation by maintaining the expression of Mcl-2. Inhibition of NF-κB increases HSC apoptosis by up-regulation of the JNK pathway. Thus, the activation as well as the induction of senescence/ apoptosis of HSC/MF are normal wound healing mechanisms that promote the establishment of

**3.2. Hepatic stellate cells (HSC) & portal myofibroblasts (MF)**

84 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

SEC's are specialized endothelial cells that lie flat in the liver sinusoids along and in direct contact with the hepatocytes. Through their membranes and specialized pores or fenestra passes high concentrations of metabolites, proteins and other blood compounds, traffic which is regulated by the size of the fenestra. SEC's play a critical role in immune-activation, rolling of T cells, macrophages and PMN migration. Liver sinusoidal endothelial cells may be affected with age and obesity. SEC from old individuals have impaired and reduced expression of VEGF likely due to impaired nuclear transport of P-STST3 and P-CREB transcription factors [82, 83]. In a rodent model of sepsis, endothelial nitrogen oxide synthase (eNOS) deficient mice and aging mice had the same mortality and mitochondrial dysfunction upon the isolation of SEC mitochondrion [84]. In obesity and during early fibrogenesis, SEC lose their fenestra, decreasing the exchange of metabolites and increase the secretion of several basement membrane components (type IV collagen, perlecan, entactin and laminin) [64]. Authors concluded that an endothelial base-line dysfunction in the aging animal is manifested by a weakened antioxidant response and inappropriate energy production from mitochondrial dysfunction due to a tipped-balance of the SEC oxi-redox systems when exposed to additional stress. This is seen in the obese towards a state of energy depletion and cellular death, apoptosis or activation of a pro-coagulant/pro-fibrogenic phase. The changes of SEC's with aging may limit O<sup>2</sup> delivery and availability to liver cells with its potential effects on mitochondrial function, a pro-fibrogenesis state and the promotion of insulin resistance status. Changes exaggerated in obesity, implying obesity may promote accelerated SEC aging processes. Interestingly, endothelial cellular senescence was inhibited in vitro and in the rodent by the activation of the liver x receptor (LXR), a nuclear receptor involved in the control of hepatic lipid and cholesterol metabolism [85]. Furthermore, LXR has been shown to play an important role in glucose metabolism, cytokine production and anti-inflammatory response.

Three types of SEC's co-exist in the normal liver sinusoid: mature SEC, SEC progenitors and bone marrow-derived SEC progenitors [86]. Mature SEC are gatekeepers of fibrogenesis by maintaining HSC in their inactivated state. SEC's regulate sinusoidal blood flow through their action on HSC and thus keep a low portal pressure [86]. In addition, mature SEC's have the largest endocytic capacity in the body fulfilling their dual cell clearance capacity (from the arterial/systemic and portal/gut systems). The liver endocytic function has been implicated in a liver-renal axis where the lack of SEC-stabilin-2 receptors inhibit the clearance of toxic molecules that manifest with mild liver fibrosis without liver dysfunction but with renal glomerular fibrosis. Not only do SEC's have many glycoproteins that serve as receptors for bacterial epitopes but as receptors for immunemodulation and pro-coagulant activity. The above mentioned SEC functions are at least partially lost at the time of sinusoid capillarization [86]. SEC capillarization is characterized by the disappearance of the fenestrae, development of a basement membrane and the appearance of characteristic markers. This phenomenon happens in chronic liver injury and it precedes activation of HSC and sequestration of macrophages. The angiogenesis process that follows is mediated by VEGF, an angiocrine response that drives neo-vessel formation in direct proportion to the degree of the sinusoidal pressure gradient. Furthermore, SEC pseudo-capillarization refers to changes that occur in endothelial cells associated with aging and senescence. It is manifested by a decrease of up to 50% of their fenestrae, development of a patchy basement membrane and partial SEC dysfunction [86]. Chronic exposure of high fat diet may accelerate aging/senescence of SEC, endothelial dysfunction with recruitment of systemic immune cells and activation of Kupffer cells inducing HSC into a fibrogenic state followed by an angiocrine response that decreases hepatic blood flow, O<sup>2</sup> delivery, and clearance of toxic molecules. As metabolic stress of neighbor hepatic cells already in mitochondrial distress due to fat accumulation progresses, a constant and growing inflammatory mielue enhances tumor development, immune-recognition failure and malignant cell expansion.

adhesion molecules such as ICAM-1, and cytokines as TNF-α, IL-1, IL-6, MIP1α, TGF-ß and RANTES. Activation that can only lead to antigen presenting, cell to cell communication and amplification and enrichment of the microenvironment with ROS promoting subsequent parenchymal cell apoptosis/necrosis. Natural Killer (NK) and CD8+ T cells developed a specific signature in livers with NASH from mice under HFD [88]. The depletion of CD8+ T cells protected murine from NASH progression but not from weight gain. In addition, NK T-cells in the liver expresses markers that recognize lipid antigen CD1d [9]. Liver NK cells undergo Thymus clonal double deletion but are positive for CD3 and CD56 and they were thought to be CD1d independent. Nevertheless, hepatic antigen-presenting cells may introduce microbial glycolipid antigens to NK cells, stimulating secretion of Th1 or Th2 cytokines which subsequently initiates an adaptive response. Hepatic NK cells have as well the ability to secrete osteopontin and sonic hedgehog, molecules known to promote the transition from NAFLD to NASH [9]. The most accepted hypothesis, continuous cell parenchymal damage and necrosis adds to a chronic inflammatory environment a dysregulation of the cell cycle regenerative process rendering tandem mutations and thus malignant cells was challenged [89]. On the NEMO knockout mouse, authors were able to develop HCC through a death receptor-independent FADD signaling pathway. Nevertheless, it wasn't until recently that the link between a metabolic hostile microenvironment, immune-recognition failure and HCC presence was established [90]. The enrichment of linoleic acid in the cirrhotic microenvironment of NASH patients promotes disruption of mitochondrial function in a greater proportion than other fatty acids as palmitic acid. Since CD4(+) T lymphocytes have a larger mitochondrial load than CD8(+) T lymphocytes, they not only generate more mitochondrial derived ROS but CD4(+) cells may undergo larger selective loss of mitochondrial function and viability. Therefore, disruption of mitochondrial function by linoleic acid mediates selective loss of intrahepatic CD4(+) T lymphocytes, status associated with HCC presence. Local metabolic changes could alter the

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immune response to a one that favors malignant cell expansion.

**3.5. Extracellular matrix (ECM)**

In the obesogenic environment, aberrant activation of immune cells has emerged as key features of the metabolic syndrome. The interaction between the adipose compartment and the liver tissue has been hypothesized as a critical interface for nutrient sensing and metabolic control [9]. In the rodent model, neutrophils infiltrate the adipose compartment as early as 3 days after starting a high fat diet, however its role as well as the role of basophils and eosinophil cells has not yet been clarified. Mast cells, which has been observed in increasing number have been implicated in the secretion of IL-6 and IFN-Ϫ [9]. Moreover, leptin, a hormone secreted specifically by adipocytes has been found to be increased during high fat diets and upregulated the expression of leptin receptors on NK T-cells. This regulation is time sensitive, and chronic leptin stimulation change NK cells from an inflammatory like response to a damped one, favoring at long term, in the liver and perhaps in other organs a susceptibility to low recognition of no selfcells, impaired anti-tumor surveillance and a flourishing nest of cancer. The former hypothesis finds support in the obese mice, where it was observed a switch from the normal Th1 immunoresponse to the Th17 immunorepertoire, phenotype that deteriorates autoimmunity [9].

The extracellular matrix (ECM) is formed by a non-cellular component in tissues and organs composed primarily of water, proteins and proteoglycans. Components created an intricate

Interestingly, aging endothelial cells from the fat compartment of mice was associated with adipose dysfunction manifested by ectopic (liver) fat deposition and adipose tissue fibrosis, increased adipose mitochondrial oxygen flux, altered lipid utilization, increased tissue oxidative stress and lower gene expression in visceral fat [87]. Nevertheless, and most important, these findings were associated with reduce fat tissue vascularity, reduced angiogenic capacity and endothelial dependent dilation with reduced nitric oxide (NO) bioavailability [87]. Limited oxygen mitochondrial availability contributes to the pro-oxidative older adipose tissue phenotype that can further impair both insulin action and vascular function, a key element in local and systemic insulin-resistant related metabolic syndromes. Changes that are exaggerated in obesity, implying obesity may promote accelerated aging processes in many organs.

#### **3.4. Resident liver immunocells**

The anatomical location of the liver and its dual blood supply ensures an optimal exposure of antigens to the hepatic resident immune cells not only from nutrients and GI microbiota but from systemic compartments, such as the adipose compartment. Kupffer cells in concert with NK, CD4+ T-cells, and local antigen presenting cells modulates the liver immune status. Kupffer cells constitutes 80% of the tissue fixed macrophages and 20% of the non-parenchymal cell population of a normal liver [9]. Their characteristic macrophage activity is polarized mainly in portal tracts where the antigen dynamics is higher from food and bacteria. Innate macrophages have the potential to initiate an inflammatory response of different proportions by upregulating adhesion molecules such as ICAM-1, and cytokines as TNF-α, IL-1, IL-6, MIP1α, TGF-ß and RANTES. Activation that can only lead to antigen presenting, cell to cell communication and amplification and enrichment of the microenvironment with ROS promoting subsequent parenchymal cell apoptosis/necrosis. Natural Killer (NK) and CD8+ T cells developed a specific signature in livers with NASH from mice under HFD [88]. The depletion of CD8+ T cells protected murine from NASH progression but not from weight gain. In addition, NK T-cells in the liver expresses markers that recognize lipid antigen CD1d [9]. Liver NK cells undergo Thymus clonal double deletion but are positive for CD3 and CD56 and they were thought to be CD1d independent. Nevertheless, hepatic antigen-presenting cells may introduce microbial glycolipid antigens to NK cells, stimulating secretion of Th1 or Th2 cytokines which subsequently initiates an adaptive response. Hepatic NK cells have as well the ability to secrete osteopontin and sonic hedgehog, molecules known to promote the transition from NAFLD to NASH [9].

The most accepted hypothesis, continuous cell parenchymal damage and necrosis adds to a chronic inflammatory environment a dysregulation of the cell cycle regenerative process rendering tandem mutations and thus malignant cells was challenged [89]. On the NEMO knockout mouse, authors were able to develop HCC through a death receptor-independent FADD signaling pathway. Nevertheless, it wasn't until recently that the link between a metabolic hostile microenvironment, immune-recognition failure and HCC presence was established [90]. The enrichment of linoleic acid in the cirrhotic microenvironment of NASH patients promotes disruption of mitochondrial function in a greater proportion than other fatty acids as palmitic acid. Since CD4(+) T lymphocytes have a larger mitochondrial load than CD8(+) T lymphocytes, they not only generate more mitochondrial derived ROS but CD4(+) cells may undergo larger selective loss of mitochondrial function and viability. Therefore, disruption of mitochondrial function by linoleic acid mediates selective loss of intrahepatic CD4(+) T lymphocytes, status associated with HCC presence. Local metabolic changes could alter the immune response to a one that favors malignant cell expansion.

In the obesogenic environment, aberrant activation of immune cells has emerged as key features of the metabolic syndrome. The interaction between the adipose compartment and the liver tissue has been hypothesized as a critical interface for nutrient sensing and metabolic control [9]. In the rodent model, neutrophils infiltrate the adipose compartment as early as 3 days after starting a high fat diet, however its role as well as the role of basophils and eosinophil cells has not yet been clarified. Mast cells, which has been observed in increasing number have been implicated in the secretion of IL-6 and IFN-Ϫ [9]. Moreover, leptin, a hormone secreted specifically by adipocytes has been found to be increased during high fat diets and upregulated the expression of leptin receptors on NK T-cells. This regulation is time sensitive, and chronic leptin stimulation change NK cells from an inflammatory like response to a damped one, favoring at long term, in the liver and perhaps in other organs a susceptibility to low recognition of no selfcells, impaired anti-tumor surveillance and a flourishing nest of cancer. The former hypothesis finds support in the obese mice, where it was observed a switch from the normal Th1 immunoresponse to the Th17 immunorepertoire, phenotype that deteriorates autoimmunity [9].

#### **3.5. Extracellular matrix (ECM)**

Three types of SEC's co-exist in the normal liver sinusoid: mature SEC, SEC progenitors and bone marrow-derived SEC progenitors [86]. Mature SEC are gatekeepers of fibrogenesis by maintaining HSC in their inactivated state. SEC's regulate sinusoidal blood flow through their action on HSC and thus keep a low portal pressure [86]. In addition, mature SEC's have the largest endocytic capacity in the body fulfilling their dual cell clearance capacity (from the arterial/systemic and portal/gut systems). The liver endocytic function has been implicated in a liver-renal axis where the lack of SEC-stabilin-2 receptors inhibit the clearance of toxic molecules that manifest with mild liver fibrosis without liver dysfunction but with renal glomerular fibrosis. Not only do SEC's have many glycoproteins that serve as receptors for bacterial epitopes but as receptors for immunemodulation and pro-coagulant activity. The above mentioned SEC functions are at least partially lost at the time of sinusoid capillarization [86]. SEC capillarization is characterized by the disappearance of the fenestrae, development of a basement membrane and the appearance of characteristic markers. This phenomenon happens in chronic liver injury and it precedes activation of HSC and sequestration of macrophages. The angiogenesis process that follows is mediated by VEGF, an angiocrine response that drives neo-vessel formation in direct proportion to the degree of the sinusoidal pressure gradient. Furthermore, SEC pseudo-capillarization refers to changes that occur in endothelial cells associated with aging and senescence. It is manifested by a decrease of up to 50% of their fenestrae, development of a patchy basement membrane and partial SEC dysfunction [86]. Chronic exposure of high fat diet may accelerate aging/senescence of SEC, endothelial dysfunction with recruitment of systemic immune cells and activation of Kupffer cells inducing HSC into a fibrogenic state followed by an angiocrine response that decreases hepatic blood flow, O<sup>2</sup> delivery, and clearance of toxic molecules. As metabolic stress of neighbor hepatic cells already in mitochondrial distress due to fat accumulation progresses, a constant and growing inflammatory mielue enhances tumor development, immune-recognition failure and malignant cell expansion. Interestingly, aging endothelial cells from the fat compartment of mice was associated with adipose dysfunction manifested by ectopic (liver) fat deposition and adipose tissue fibrosis, increased adipose mitochondrial oxygen flux, altered lipid utilization, increased tissue oxidative stress and lower gene expression in visceral fat [87]. Nevertheless, and most important, these findings were associated with reduce fat tissue vascularity, reduced angiogenic capacity and endothelial dependent dilation with reduced nitric oxide (NO) bioavailability [87]. Limited oxygen mitochondrial availability contributes to the pro-oxidative older adipose tissue phenotype that can further impair both insulin action and vascular function, a key element in local and systemic insulin-resistant related metabolic syndromes. Changes that are exaggerated in obesity, implying obesity may promote accelerated aging processes in many organs.

86 Hepatocellular Carcinoma - Advances in Diagnosis and Treatment

The anatomical location of the liver and its dual blood supply ensures an optimal exposure of antigens to the hepatic resident immune cells not only from nutrients and GI microbiota but from systemic compartments, such as the adipose compartment. Kupffer cells in concert with NK, CD4+ T-cells, and local antigen presenting cells modulates the liver immune status. Kupffer cells constitutes 80% of the tissue fixed macrophages and 20% of the non-parenchymal cell population of a normal liver [9]. Their characteristic macrophage activity is polarized mainly in portal tracts where the antigen dynamics is higher from food and bacteria. Innate macrophages have the potential to initiate an inflammatory response of different proportions by upregulating

**3.4. Resident liver immunocells**

The extracellular matrix (ECM) is formed by a non-cellular component in tissues and organs composed primarily of water, proteins and proteoglycans. Components created an intricate scaffold where organ cells get structural support with a dynamic and continuous traffic of water, ions, metabolites, proteins and cells on passant to maintain organ physiology. As such, ECM interactions with organ cell components regulate cell differentiation, adhesion, proliferation, migration and survival [64]. The collagen family is the major fibrillar proteins of the ECM and the body (approx. 30% of the total protein contain) [64]. There are three main classes of collagen, fibril-forming which include types I, II, III, XI, XXIV and XXVII the most common varieties and their role is mainly mechanical by conferring tensile strength to both tissue and organs. Fibrilassociated collagens with interrupted triple helix (FACIT's) includes type IX, XII, XIV, XIX, XX, XXI and XXII; this subclass of proteins do not form fibrils themselves but bind to the surface of pre-existing collagen favoring fibril enlargement. Finally, type III collagen serves as anchoring collagen between the epithelial cells and the lamina reticularis constituting the basement membrane where type IV collagen is most abundant. Non-collagenous proteins include fibronectin, tenascin, laminins, fibrillins and matrix-cellular proteins. While the former peptides play a major role in cell differentiation, cell growth, adhesion and migration, matrix-cellular proteins, i.e. thrombospondin-1 and 2, osteonectin, osteopontin and cyr-61/connective tissue growth factor (CTGF) serve mainly as a vehicle for cell signaling. Proteoglycans are carbohydrate enriched proteins which retain large quantities of water regulating the smooth trafficking of molecules to and from the cell with numerous signaling active sites for growth factors.

IV where oxygen is reduced to form H<sup>2</sup>

O2

) and hydroxyl radical (OH−

gen peroxide (H<sup>2</sup>

O. The electron transport process is coupled to a proton

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) which are mostly produced because electron

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pumping process creating a proton gradient between the mitochondrial membranes, gradient that is dissipated by Complex V (ATP synthase) through ATP synthesis. A control mechanism is created by the "proton leak", mechanism that generates heat instead of ATP [10]. Much of the leak is a catalytic reaction generated by the uncoupling proteins (UCP's) which play an important role in reducing proton gradient, heat and ROS [10]. Mitochondrial aging and senescence are linked to reduced ATP production and increase ROS production, i.e. superoxide (O2−), hydro-

leakage at the level of CI and CIII [17]. Mitochondria function benefit from the role of estrogen in plasma through its binding to the nuclear estrogen receptor that enhances a signaling to prevent oxidant stress and also inhibits the renin-angiotensin-aldosterone system [10]. Thus, sex differences in mitochondrial function may explain the disparity in overall survival between men and

The reasons why the mitochondrion conserves a cell independent genome are not clear, but it is intuitive to imply self-energy regulatory processes are united through a fine tune mechanism between energy expenditure (ATP use) and energy production (ATP synthesis) at every organelle level. It may provide an overall advantage for survival of the cell, the organ and entire biological living system. The gradual ROS response theory of aging argues a protective role of ROS in early life, when cell oxidative damage and ROS production are low; however, later in life ROS reaches a level where its beneficial effects (as the one observed in dietary restriction and/or exercise) are overcome by its detrimental effects elicited by a higher cell oxidative stress (as the one observed in high fat diet and sedentary habits) [17]. The effects that are amplified include loss of genomic controls (p53), microRNA dysregulation, loss of function of telomerase reverse transcriptase (TERT) and a lower immune-surveillance status. Although the role of p53 in the mitochondrion is not completely clear, p53 binds to the Peroxisome proliferator-activated receptor Gamma-Coactivator 1 alpha and Beta (PGC-1α and ß) fomenting their inhibition of expression and therefore downregulated oxidative function. In addition, p53 target p16 and p21, factors that triggers G1-phase cycle arrest by inhibiting cell cycle regulatory kinases Cdk4 and Cdk2 [17]. The third known effect of p53 at the mitochondrion level is to promote cell apoptosis by increasing mitochondrial membrane permeability with leakage of cytochrome proteins, a direct activator of the caspase cascade. The function of TERT is highly affected by levels of ROS production and its protective patterns are only observed with low ROS levels. The role of microRNA in the mitochondrial environment remains to be elucidated. The Mitochondrial Free Radicals Theory of Aging (MFRTA) has been the most popular theory to explain the cell aging process where increasing production of mitochondrial ROS with lower ATP production are the main factors responsible for cell aging and corresponding mitochondrial ultrastructure changes [17, 91]. As mentioned, leakage of electrons at the level of CI and CIII transfer are larger with age and the higher potential for DNA damage. 8-oxo-7.8-dihydro-2′deoxyguanosine (8-oxodG) is one of the most abundant DNA mutations caused by oxidative conversion to guanosine. Furthermore, its accumulation follows an inverse and exponential curve against life expectancy in several mammals [17]. Recently, it was described that humans with longer longevity have a higher content of mitochondrial DNA (mtDNA) per cell in different organs, and support the notion of ethnic background on mtDNA influence and life span. The frequency of mtDNA mutations occurs at different rate depending on the organ. Skeletal and

women, differences that may be taken into account during animal models studies.

The ECM continuous remodeling is a complex process that integrates proteins and cellular components from local and distal environments [64]. The degradation of ECM proteins are closely controlled by matrix metalloproteinases (MMP's), a superfamily of zinc-dependent endopeptidases highly regulated by specific inhibitors such as the tissue inhibitor of metalloproteinases (TIMP's). In the liver, cellular component involved in collagen synthesis and deposition included HSC, MF and vascular smooth muscle cells [64]. In chronic liver injury, an override mechanism of collagen deposition regulation promotes massive ECM expansion. The characteristic features of abnormal liver fibrogenesis as a consequence of continuous liver injury and activation of collagen secreting cells include damage to the epithelial/endothelial barrier, recruitment of inflammatory cells, secretion of cytokines and other inflammatory mediators, further generation of ROS, progressive deposition of collagen with expansion of ECM and worsening organ fibrosis and subsequent metabolic changes of portal hypertension.
