AMBIENTES GEOTECTÓNICOS DE GENERACIÓN DE MAGMAS

CHC is associated with elevated expression of a number of biomarkers of cellular senescence, leading researchers to suggest that HCV infection may result in an aged phenotype. CHC is associated with increased mortality from both hepatic and extra-hepatic disease suggesting increased susceptibility to disease (Lee et al., 2012) – a recognised consequence of the normal ageing process. Ageing itself also appears to have a negative impact on the history of HCV infection (chapter 1.3.2.1); older patients experience more rapid disease progression and a poorer response to IFN-based treatment (Hoare et al., 2010a). Additionally, the

morbidity and mortality associated with HCV infection increases exponentially with age (Grebely and Dore, 2011).

1.6.3.1 Cellular senescence in CHC

CHC is associated with telomere shortening in numerous cell types (Kitay-Cohen et al., 2008, Sekoguchi et al., 2007). In chronic liver disease, hepatocyte cell cycle turnover results in hepatocyte telomere shortening and induction of replicative senescence (Wiemann S, 2002), linked to progressive fibrosis and cirrhosis. Moreover, chronic inflammation is associated with the generation of

ROS with attendant effects upon telomere length. Sekoguchi et al. (2007) demonstrated that active HCV infection was associated with hepatocyte telomere shortening, but that this signal was lost in patients with normal ALT levels. Telomere shortening also correlated with lower platelet counts and higher levels of serum hyaluronic acid, markers of increased hepatic fibrosis. As Sekoguchi and colleagues (2007) were unable to identify hepatocytes with

evidence of oxidative damage independent of telomere length, the investigators proposed that the primary cause of cellular senescence in CHC was telomere shortening due to cell turnover. They also observed more hepatocytes

demonstrating morphological features of senescence suggesting the rate of senescence is increased in CHC (Sekoguchi et al., 2007). Although oxidative stress was not considered the main mechanism of cellular senescence, it was postulated to contribute to accelerated telomere shortening in advanced fibrosis.

Interestingly, Hartmann et al. (2011) demonstrated that telomerase gene mutations were more frequent in cirrhotic patients than in healthy controls, or HCV-infected patients with indolent disease, leading them to postulate that telomere shortening may represent a causal factor accelerating fibrosis and impairing liver regeneration in response to chronic liver injury.

1.6.3.2 Immunosenescence in CHC

Both HCV and ageing are associated with effects on the immune system. Chronic immune activation may result in the development of T cells that are capable of cytokine secretion yet demonstrate failure to proliferate. CD57 is a cell marker commonly expressed on such T cells and correlates with senescence (Kern et al., 1996). Manfras et al. (2004) found that increased oligoclonality and expression of CD57 on peripheral lymphocytes was associated with increased levels of fibrosis and reduced response to treatment in patients with HCV infection, perhaps explaining the poorer treatment outcomes classically seen in older patients (Sherlock, 1995).

HCV-infected individuals also have shorter lymphocyte telomeres (characteristic of immunosenescence) in comparison with healthy controls, and subjects in whom HCV has been eradicated (Kitay-Cohen et al., 2008, Grady et al., 2016). In contrast to the findings in hepatocytes, Kitay-Cohen and colleagues (2008)

demonstrated similar cell turnover in HCV-infected and healthy individuals, suggesting lymphocyte telomere shortening is not a result of shorter cell cycles. They also demonstrated that rates of aneuploidy persisted despite normalisation of ALT, suggesting that genomic instability secondary to telomere shortening endures despite disease remission (Kitay-Cohen et al., 2008).

Chronically HCV-infected individuals have reduced CD4 T lymphocyte telomere lengths compared to healthy controls (Grady et al., 2016). Hoare and colleagues (2010b) also demonstrated this association, and estimated the overall difference in telomere length to be equivalent to 10 years additional ageing. Furthermore, they showed that CD4 T lymphocyte telomere length correlated with predictors of morbidity and mortality (increased fibrosis stage, increased portal tract inflammation, prolonged PT and increased bilirubin) and suggested that

individuals with mild disease but shortened telomeres should be offered antiviral therapy at the earliest opportunity.

Reduced hTERT mRNA expression, which is reflective of telomerase activity, has been reported in the peripheral lymphocytes of patients with HCV infection, and is unrelated to activity or stage of disease. Reduced telomerase activity

represents premature ageing, and Satra and colleagues (2005) have suggested that deregulated hTERT mRNA expression may be involved in the

immunopathogenesis of HCV infection. In contrast to this, Hoare et al. (2010a) were unable to demonstrate a failure of telomerase induction in peripheral lymphocytes of patients with CHC.

1.6.3.3 INK4/ARF regulation in HCV

CKDN2A has been suggested as a potential BoA, expression of which correlates with ageing in a number of animal tissues (Herbig et al., 2006, Nielsen et al., 1999, Zindy et al., 1997) (chapter 1.6.1.3). CDKN2A accumulates with ageing in haematopoetic stem cells, and CDKN2A inhibition is associated with reduced apoptosis and improved stress tolerance (Janzen et al., 2006). Robinson and colleagues (2013) observed higher expression of CDKN2A mRNA and the related transcripts ARF and CDKN2B in the PBMCs of chronically HCV-infected subjects compared with matched controls, and found that expression of CDKN2A and ARF mRNA correlated with disease stage. Elevated expression of CDKN2A mRNA correlated with increased p16 protein levels. They also demonstrated a trend

towards shorter telomere length in the PBMCs of patients with CHC compared with healthy controls but this failed to reach statistical significance.

1.6.3.4 Oxidative stress in HCV

Conditions causing a mismatch between production and removal of ROS lead to a change in the redox state of the cell, known as oxidative stress. Oxidative stress promotes cellular senescence both directly and indirectly through effects on telomere function. Mitochondria are an important source of ROS, and it has been postulated that HCV may contribute directly to mitochondrial dysfunction

(Finkel, 2011).

HCV viral proteins, notably core and NS3/4A, localise to the OMM (Kasprzak et al., 2005, Horner et al., 2011). A number of in vitro studies have reported an increase in ROS production and mitochondrial dysfunction in association with HCV proteins, particularly the core protein (Korenaga et al., 2005, Okuda et al., 2002). Korenaga et al. (2005) demonstrated that incubation of core protein with control mitochondria caused glutathione oxidation and increased ROS

production. These changes are thought to relate to enhanced mitochondrial uptake of calcium ions (Korenaga et al., 2005, Piccoli et al., 2007, Quarato et al., 2013).

Barbaro et al. (1999) found a higher prevalence of ultrastructural alterations of mitochondria in Gt1b infected patients together with depletion of mitochondrial DNA, and postulated that impaired oxidative phosphorylation in these patients may promote hepatotoxicity.

Quarato and colleagues (2013) proposed the existence of a positive feedback

loop with increased Ca2+ _{flux from the ER into the mitochondria leading to}

increased ROS production and enhanced opening of the mitochondrial

permeability transition pore (MPTP). Activation of the MPTP leads to further

alteration of the redox state with subsequent effects on ER-mitochondrial Ca2+

homeostasis. Opening of the MPTP results in depletion of the low molecular weight molecules (carnitine, coenzyme A (CoA) and NAD+) required to import long chain acyl-CoA for β-oxidation. Accumulating acyl-CoA molecules are thus forced towards triglyceride synthesis leading to hepatic lipid deposition and steatosis, with consequent effects on fibrosis progression (chapter 1.3.2.2).

Differing levels of oxidative stress appear to lead to different pathogenic outcomes; low production of ROS reportedly favours pro-survival adaptation, whereas high levels of ROS lead to permanent opening of the MPTP and resultant osmotic swelling, rupture of the OMM and ultimately, cell death.

1.6.3.5 Lifestyle factors

Studies have demonstrated a high prevalence of various risk behaviours amongst HCV-infected individuals. For instance, Zani et al. (2009) gathered self-reported data on smoking and alcohol use amongst 229 patients attending a clinic in Italy. Over half of the male patients consumed alcohol and around 44% were current smokers. Of the female patients, approximately 35% consumed alcohol and 27% were current smokers. Additionally, PWIDs make up the majority of the HCV- infected population in the UK (HPS, 2015).

Shorter telomere length has been reported in association with alcohol use, substance misuse and smoking (Pavanello et al., 2011, Song et al., 2010, Yang et al., 2013). Smoking also accelerates the age-dependent accumulation of

cytogenetic abnormalities in lymphocytes (Ramsey et al., 1995). Grady et al. (2016) found that HCV-infected PWIDs had significantly reduced CD4+ T lymphocyte telomere lengths in comparison to healthy controls (Grady et al., 2016). This accumulation of DNA damage and accelerated cellular senescence (Song et al., 2010) may lead to faster disease progression and HCC development in individuals with HCV (chapter 1.3.2.2).