Frontline

Ageing is a more universal phenomenon than once thought. Even unicellular organisms such as bacteria and yeast do senesce (Aguilaniu et al. 2003; Ackermann et al. 2003). It is therefore rather tempting to speculate that multicellular ageing may have evolved from unicellular ageing. I am interested in studying the evolutionary forces that drive unicellular ageing, hoping that the same forces operate in the ageing process of more complicated forms of life. Unicellular ageing has been attributed to asymmetric segregation of damaged macromolecules at mitosis. If one of the daughter cells that arise from cell division contains, as a result of asymmetric inheritance of maternal damage, more damage (i.e. is functionally older) than the other, the population will evolve into a heterogeneous mixture of old and rejuvenated cells. With sufficient asymmetry and sufficiently low rates of damage accumulation, the population may continue to grow and survive, owing to the rejuvenated cell arising from each division. Otherwise, the population reaches clonal senescence.

Mathematical modelling is a promising alternative to experimental evolution studies because the latter are time-consuming and the experimenter needs to have the environment tightly under control and have all relevant information about the intra-organismal events as well as the ecological conditions that may influence the evolution of the phenomenon of interest. These problems are particularly relevant in studying the evolution of asymmetric damage segregation because experimental data are currently scarce. I use theoretical modelling to investigate the evolution of asymmetric segregation of non-genetic macromolecular damage, e.g. oxidised proteins.

One should account for both extrinsic (environmental changes) and intrinsic noise that accompany the evolution of asymmetry. Intrinsic stochasticity in segregation of damage arises from the reduction of damage entities in the cell following the aggregation of damaged proteins. Damaged particles that form an aggregate are not free to choose the daughter cell to segregate to. Rather, all particles in an aggregate have the same fate, i.e. the aggregate's fate. This phenomenon introduces substantial noise to the system that can be accounted for only by a stochastic model. The literature is lacking such models at present. Deterministic models have suggested that asymmetric damage segregation allows the lineage to withstand higher levels of damage before entering clonal senescence (Erjavec et al, 2008). However, due to the omission of stochastic effects, such models have not been able to explain all experimental observations.

Using stochastic models, I have determined the evolutionary forces that drive the evolution of asymmetry (Figure 1). Specifically, high rates of damage, the severity of damage, and slow proliferation rates promote higher levels of asymmetry. The results help us determine the contributions of individual asymmetry determinants, both environmental and organismal. In particular, environmental stress favours cells with more asymmetric segregation strategies. One interesting prediction is that the yeast Saccharomyces cerevisiae (S. cerevisiae), which produces at division an old mother cell and a rejuvenated daughter cell (Haber 2003), has probably evolved in harsher environments that Schizosaccharomyces. pombe (S. pombe), which has almost symmetric distribution of damage between the daughter cells (Minois et al. 2006).

Figure 1. Due to limited resource availability, there is a trade-off between investments in reproduction and maintenance/repair, and the model predicts that investment in either function acts to reduce the selection pressure for asymmetry. Since damage accumulation is the product of the incoming stress and the strength of cellular defence/repair systems, the impact of alterations in the environmental stress on selection pressure will be major and dominant.

Evolution of multicellularity is known as one of the major evolutionary transitions. A unicellular organism is directly exposed to the environment, and unless the environment changes on rapid time scales, it does not evolve complex signalling pathways to respond to environmental changes. In a multicellular organism, however, a cell experiences the microenvironment which before the evolution of advanced homeostatic systems may have well been exposed to transient perturbations (even with constant external environment surrounding the whole organism). Therefore, multicellular organisms need intracellular signalling pathways capable of responding to both environmental and micro-environmental cues. The other complication at the transition to multicellularity is that such cellular responses need to be tuned for the well-being of the organism, rather than the particular cell. I am trying to see how asymmetric damage segregation is modified as multicellularity evolves. Insights into the events that have occurred at this transition may then help proceed to humans.

The talk that I gave at the 2008 Annual Scientific Meeting "Ageing: Molecules to Man" in Brighton showed the results of my work during the first year of my PhD. I am very interested in the relationship between Non-Alcoholic fatty liver disease and ageing. For a long time it was considered, that the liver is almost unaffected by ageing and age-related disease. However, more recently several functional and morphological changes have been observed in the liver with age, like reduced blood flow and liver mass and a very important change: the reduced ability to regenerate.

The modern lifestyle characterised by less exercise and excess food intake is the main contributor to obesity in western countries. These factors are not only responsible for the onset of diseases such as diabetes and cardiovascular complications, but also for metabolic syndrome and Non-Alcoholic Fatty Liver Disease (NAFLD). NAFLD is the term used for the liver disorder similar to alcoholic liver disease, which is observed in patients with the metabolic syndrome and is characterized by increased accumulation of triglycerides in the hepatocytes (hepatic steatosis). Fibrosis, an advanced stage of NAFLD, is the result of chronic liver injury due to alcohol intake, drugs, infections (hepatitis C virus [HCV]) or metabolic disorders. Normally it evolves over decades, but can progress over weeks/months in some patients such as recurrent HCV infection in immunocompromised patients post liver transplantation. In all fibrotic reactions, the underlying cellular and molecular mechanisms involve leukocyte infiltration, persistence of inflammation in the tissue and proliferation of cells with a myofibroblast phenotype. Fibrosis can be considered as a wound-healing response that has become excessive due to a failure of the liver to degrade the excess of extracellular matrix and scar tissue appropriately, which eventually leads to adverse effects on liver function.

In liver an increase in SA-β-Gal activity (marker of cellular senescence) has been observed in liver cirrhosis, which is the end stage of chronic liver disease. This is consistent with the hypothesis that senescence is associated with liver disease. Also, an increase in senescent cells with age has been described in various tissues in mammals. To determine the effect of senescence on liver ageing we measured γ-H2A.X foci frequency in hepatocytes from mice at 4 different age groups (12, 22, 36 and 42 month old). Our results show a significant increase in the frequency of γ-H2A.X positive hepatocytes in liver with mouse age. Then, we decided to look in more detail at the spatial disposition of the γ-H2A.X positive cells. We found that γ-H2A.X positive cells could be found predominantly in specific areas of the liver.

4-Hydroxy-2-Nonenal (HNE) is one of the major aldehydic metabolites of lipid peroxidation and the most reliable marker of lipid peroxidation. HNE may directly activate hepatic stellate cells and lipid peroxidation may play a role in hepatic fibrogenesis. Increased HNE adducts have been reported in liver tissues from animals with experimental iron overload and in patients with different chronic liver diseases. Since oxidative stress has been shown to play a role in cellular senescence and in liver disease in both humans and mice, we decided to look at HNE prevalence in mouse liver with age. We observed an age-dependent increase in HNE adducts with age. We also found an age-dependent increase in alpha-SMA which is a marker of activated hepatic stellate cells (HSCs). The initial activation of HSCs is likely to be a result of stimuli produced by neighbouring cells namely hepatocytes, Kupffer cells, circulating leukocytes, platelets and sinusoidal endothelial cells in response to liver injury. Such stimuli include reactive oxygen species (ROS) / lipid peroxides, growth factors and inflammatory cytokines. It could be that increased lipid peroxidation with age in mouse liver, as seen using HNE, is an important stimulus for the activation of HSCs and finally increased liver fibrosis. Another possibility is that senescent hepatocytes themselves contribute to activation of HSCs. It has been well documented that senescent fibroblasts overexpress proteins that remodel the extracellular matrix or mediate local inflammation, altering the surrounding microenvironment. Moreover, our group has shown that senescent cells produce high levels of ROS. It is possible that these factors together could contribute to the activation of HSCs and the onset of liver fibrosis. However, our results only show a correlation between these factors and no causality has yet been established.

Sandra A. Jones^‡, Richard D. Walton & Matthew K. Lancaster.

^‡University of Hull, Hull, HU6 7RX; University of Leeds, Leeds, LS2 8JT.

Progressive ageing is unfortunately associated with an increasing risk of developing health problems. Statistically one of the most common medical conditions afflicting the elderly is heart disease,[1] second only to arthritis as the most prevalent medical condition affecting the elderly.  Much is known, discussed, and broadly acted upon, regarding the many modifiable risk factors for heart disease such as blood pressure, cholesterol, weight and smoking, however the largest risk factor for heart disease is not normally referred to - increasing age.  In our opinion this is perhaps because this risk factor is considered un-modifiable and unavailable for commercial exploitation - yet.  However there may be opportunities on the horizon and perhaps further consideration should be given to how to combat this dominating risk factor for cardiovascular problems.

Heart disease is a broad term frequently used to cover all dysfunction of the cardiovascular system.  However many changes occur in the cardiovascular system of elderly individuals in a sub-clinical but progressive manner which are more commonly associated with pathology.  This sub-clinical accumulation of defects appears to progress steadily throughout the lifespan until they become sufficient to produce clinical dysfunction and symptoms leading to a diagnosis of heart disease.[2] Such changes include both structural remodelling and functional alterations. 

Problems of the Ageing Cardiovascular System

Common problems of the cardiovascular system afflicting the elderly are (Figure 1):

• ‘Hardening' of the arteries due to calcification and remodelling of the arterial wall. This leads to increased vascular resistance, increased blood pressure and an increased work load on the heart.[3, 4]
• Decreased relaxation and an increased stiffness of the heart muscle itself. Remodelling of the extracellular matrix of the heart is associated with reduced compliance limiting cardiac capacity, decreasing maximal output and again increasing the workload on the heart.[5, 6]
• Impaired coronary circulation. Atherosclerotic plaques accumulate in the arteries of the cardiovascular system including those supplying the heart with blood in an age and other risk-factor dependent manner (particularly associated with cholesterol).[7] As they enlarge and/or rupture causing clots to form the oxygen supply to the heart is compromised and cardiac damage is likely to occur. Increasingly people survive such heart attacks, but the diminished function of the damaged heart often means the remaining life of these individuals is associated with significantly curtailed exercise capacity.
• Impaired peripheral circulation. What affects the coronary arteries also affects all other arteries and atherosclerotic plaques accumulate in the peripheral vasculature with age. When plaques enlarge or rupture creating clots in the limbs or cerebral vasculature the associated limb or organ is in danger. Intermittent claudication is pain in the limbs normally associated with exertion in the elderly but due to impaired circulation to a limb causing in-sufficiency in oxygen supply.[8] The associated intolerance to exercise contributes significantly to immobility in the elderly and can lead to loss of limbs in a worst case scenario. Similarly if atherosclerosis affects the cerebral vasculature stroke is likely with its associated problems and diminished oxygen supply to the brain is also associated with an early onset of dementia and other disorders of neural function.
• Pacemaker dysfunction. The normal pacemaker of the heart, the sinoatrial node shows an age-dependent deterioration in its ability to operate at high frequencies and to control the beating rate of the heart. The majority of pacemaker dysfunction occurs in the elderly increasing in incidence in an age-dependent manner. Untreated this condition predisposes to fainting, cardiac arrhythmias, stroke, sudden death and exercise intolerance.[9] With age the sinoatrial node progressively loses proteins required for the heart beat to be initiated and for this electrical signal to propagate down the heart causing normal activation.[10, 11] This inactivation and disconnection of the hearts own pacemaker requires treatment by implantation of an artificial one and over 70% of such implants are made in those over 65 years of age.[12]
• Inability to respond to stress. Cardiac capacity has therefore fallen, the work load on the heart has increased and the ability of the pacemaker to provide a high activation frequency has fallen so it is no surprise to find the heart cannot respond to stress very well. The normal response to cardiovascular stress such as exercise is to release nor-adrenaline and adrenaline which acts on adrenergic receptors on the heart causing the contractile strength to increase and the heart rate to accelerate. In the aged heart however this response is significantly blunted limiting the ability to respond to stress.[13, 14] Key factors behind this seem to be a desensitisation of the adrenergic receptors on the heart and a decline in the ability to generate the normal signal associated with stimulation of these receptors.
• Increased susceptibility to damage. The aged heart is more likely to be subjected to ischemia or increased operational stresses due to the changes outlined above but unfortunately with age the heart loses its ability to resist damage in the event of such stresses. Comparable levels of ischemia, hypoxia, or even simple increases in stimulation by adrenaline, which can be tolerated by the young heart can cause considerable damage in the old heart causing cell death or disorganised activation of the heart (arrhythmias).[15] This appears to be due to a loss of the protective arrangements that guard the heart from damage although the precise mechanisms are still controversial. Changes in heat shock proteins, mitochondria and ion channels are all implicated in this age-associated decline in tolerance but the precise interplay and how this may change with age is still being worked out.

Following this sobering list of reasons why the aged cardiovascular system is likely to become a dysfunctional cardiovascular system the natural question arising is how can we intervene to improve the outlook for those old at heart?  Much has been written regarding the benefits of modifying a variety of risk factors, e.g. high blood pressure and cholesterol, and how these can limit the risk of heart attack.  However whilst these interventions can prevent problems associated with ischemia and blockage of the vasculature avoiding a test of the weakened ability to resist damage what influence do they have on the background problem of ageing of the cardiovascular system?  Changes such as the development of pacemaker dysfunction, intolerance and inability to respond to stress, and a decline in the maximal cardiac output appear to occur whether you follow a healthy diet or not, so what should we do to tackle the underlying issues of the ageing cardiovascular system?

Preserving Healthy Function in the Ageing Cardiovascular System

Exercise is frequently advocated as a way to improve cardiovascular health and for several key areas there is plenty of evidence suggesting this is true.  Exercise beneficially modifies cholesterol profiles potentially limiting arthrosclerosis,[16] it can help lower blood pressure and weight,[17] limiting baseline cardiovascular stress, and it improves the heart's ability to withstand acute stress such as ischemia.[18]  As such regular exercise increases the likelihood of living to an older age extending mean lifespan (although not maximal lifespan),[19] but what direct effects does it have on the ageing of the heart?

Here despite some studies showing that exercise can prevent age-associated changes in gene expression within the heart the evidence becomes slightly disappointing.[20]  Masters athletes who have trained intensively throughout their life, still experience a fall in maximal achievable heart rate, and maximal cardiac output.[21]  Although there is evidence that this fall is not as rapid as that seen in sedentary individuals.  A more surprising finding is that people who have performed intensive endurance exercise throughout their life (e.g. competitive marathon runners and cyclists) are actually more likely to suffer from cardiac pacemaker dysfunction and arrhythmias and require an artificial pacemaker in later life than those who have lead a relatively sedentary existence [22, 23], leading us to question whether exercise (or certainly intensive exercise) is an ideal solution for healthy ageing of the heart?

So what other solutions are there for an ageing cardiovascular system?  Diet is frequently considered as an important modifier of cardiovascular stress and the atherosclerotic process.  By keeping salt low in the diet blood pressure and hence cardiovascular stress can be limited.  Cholesterol has become one of the most popular modifiable risk factors for atherosclerosis to be targeted by a variety of campaigns in recent times.  It is now well established that keeping cholesterol low associates with slower development of atherosclerosis and reduced incidence of coronary heart disease and the widely-prescribed statin family of drugs target this relationship (although arguably not as well as diet can - but with less effort).  Much remains to be characterised regarding the formation of atherosclerotic plaques though and the precise blood lipid components posing the greatest danger or benefit, with debates continuing regarding the relative use of measures of LDL (low-density lipoproteins), HDL (high-density lipoproteins), their sub-components, and their ratio as predictors of heart disease.  One thing that is clear though is that low LDL and high HDL cholesterol are generally optimum for preventing atherosclerosis.

As a separate consideration of diet there is a tendency for the elderly to consume a diet of poorer nutritional quality and suffer from deficiencies of some vitamins and minerals.  Much has been made of the potential benefits of vitamin E or C supplementation to preserve cardiovascular function in the elderly and guard against the potential oxidative stress of ageing or even cardiac ischemic damage.  Such supplementation trials and studies however show very mixed success and data with potential beneficial effects in populations that may have already had deficiencies but unfortunately there is no clear sign that large anti-oxidant vitamin supplementation or consumption will prevent cardiovascular disease or mortality from such causes.  The more interesting area of work at the moment in this area is actually on vitamin D where a link between vitamin D deficiency and cardiovascular risk has been established.[24]  In the elderly vitamin D absorption and synthesis appears to be impaired and due to mobility issues exposure to sunlight can be reduced.  This offers therefore not only a risk factor for osteoporosis with ageing but also apparently the risk of cardiovascular disease.  The precise mechanisms why vitamin D deficiency should be so problematical for the cardiovascular system are unclear but may include reductions in blood pressure.[25]  Some studies have already questioned whether vitamin D supplementation may help healthy ageing in the elderly with mixed results, perhaps because overly-high vitamin D levels, can actually be a risk factor itself, but more are likely to follow to establish if this is a route to healthy cardiovascular ageing.

Diet also impacts body weight, itself a risk factor for cardiovascular disease, and with caloric restriction being one of the very few interventions known to consistently increase maximal lifespan a careful eye on calories consumption is perhaps warranted.  In animal models and humans on caloric restricted regimes cholesterol, bodyweight, blood pressure and resting glucose levels fall (all important cardiovascular disease risk factors) but perhaps more surprisingly cardiac tolerance to ischemia increases.[26]  As such caloric restriction offers many of the advantages of exercise for the cardiovascular system and heart itself, but encouraging participation in either is difficult!  Interestingly the effects of exercise and caloric restriction may also be additive and of benefit later in life with one study showing complete restoration of protection against ischemia in elderly animals when exercise and caloric restriction were combined.[27]

On balance it appears regular exercise and/or caloric restriction make it more likely you will get to experience more of the ageing process by reducing mortality projections (and not just death through cardiovascular-related causes) but they do not prevent ageing, and in some cases may actually increase the occurrence of age-associated cardiac issues. Much work however remains to be done to investigate the fine detail of the ageing cardiovascular system.  The recent findings that even the adult heart can have progenitor cells embedded within it capable of dividing into new cardiac cells offers new hope for renewing the ageing heart keeping it youthful in function and operation.[28]  The bad news is we appear to lose these progenitor cells as we age and they also become reticent to act, divide and repair damage in later life.  Restoration of the activity of these progenitor cells and re-establishing the heart's ability to repair itself is perhaps one of the most exciting future prospects for ensuring healthy cardiac function for all in old age.  Recent work has suggested that elevated levels of IGF-1, such as can be stimulated by exercise, are important for keeping progenitor cells active into old age and can extend lifespan.[29]

A Prescription for Healthy Ageing of the Cardiovascular System

Until we understand more about how the cardiovascular system ages and how to keep it youthful the best advice is keep blood pressure and LDL cholesterol down,[30, 31] keep vitamin D,[24] HDL cholesterol and IGF-1 up,[32, 33], keep body weight and caloric intake down and stay active with exercise most days of the week.[34]  These interventions may not prevent ageing entirely but will increase the chances of a healthy longer life (on-average) and sounds like a good excuse for regular steady 30 minute runs in the sun - a prescription which will impact many of these factors.

Figure 1: Elevated levels of senescence associated β-galactosidase activity within prematurely senescent chronic wound fibroblasts 

Currently our research activities are centred on two major areas:

Dysfunctional wound healing in the aged

Chronic wounds are most simply described as wounds that fail to heal.  They occur most frequently on the lower limbs and exist in three principal forms (pressure sores, venous ulcers and diabetic ulcers).  Chronic wounds occur in an estimated 1% of the population and their treatment is currently estimated to cost the NHS over £1 billion/year.  In chronic wounds the normal cellular responses to acute injury are impaired and the wounds are characterised by a defective wound matrix, a failure of re-epithelialisation and prolonged inflammatory response. Through our research efforts we have demonstrated that chronic wound fibroblasts (CWF) are dysfunctional with respect to their normal wound healing functions (proliferation, wound repopulation, extracellular matrix reorganisation; Cook et al., 2000; Stephens et al., 2003a; Stephens et al., 2004) compared to patient matched normal fibroblasts (NF) and that this is related to a significantly reduced proliferative lifespan and early onset of senescence compared to NF (Fig. 1; Wall et al., 2008).  However, investigation of telomere dynamics utilising Single Telomere Length Analysis has indicated that induction of senescence in CWF was telomere-independent.  Microarray and functional analysis has revealed lower expression, production and activity of several CXCL chemokines in CWF compared to NF suggesting that an inability to correctly express a stromal address code is implicated in the disease chronicity (Wall et al., 2008).  

There is increasing evidence to implicate the excessive production of reactive oxygen species (ROS), such as the superoxide radical (O₂^.-) and the highly reactive hydroxyl radical (^.OH) species, by inflammatory cells in the pathogenesis of chronic inflammatory conditions, such as periodontal diseases, rheumatoid arthritis and dermal wounds (Waddington et al., 2000; Moseley et al., 2004b).  Elevated ROS production is also implicated as a causative factor to the ageing process.  Such events result in extensive degradation of extracellular matrices and altered cellular metabolism.  We have discovered that this early onset of cellular senescence is accompanied by an increase in CWF ROS generation, which further contributes to the elevated oxidative stress within the chronic wound environment and further depletes the capacity of CWF to withstand the accumulation of ROS-induced damage (Wall et al., 2008).  We have also demonstrated that there is a lack of major enzymic antioxidant upregulation in CWF suggesting that the adaptive mechanisms of protection against oxidative stress are defective.   This links with our previous identification of a number of oxidative stress-related biomarkers, which we aim to utilise as prognostic/diagnostic biomarkers of chronic wound infection and disease activity (Moseley et al., 2004a & b).  We are currently investigating ways to counteract this oxidative stress imbalance through over expression of antioxidants and novel agents in our cell populations and through the use of biomaterial-based wound dressings.  Other investigations are centred on the use of biopolymers to protect growth factors from the harsh chronic wound environment (Hardwicke et al., 2008)

Our isolation and characterisation of chronic disease cells has driven our efforts to develop a chronic wound reporter cell line as part of our interests in the 3Rs (replacement, reduction and refinement of animals in research).  Cultures of CWF and patient-matched NF cells have been immortalized using a human telomerase containing retroviral vector.  Microarray analysis of the global gene expression profiles of the CWF and NF, both normally and in response to a wounding stimulus, have enabled the identification of disease-specific marker genes.  The promoters of these disease marker genes have been cloned into fluorescent reporter plasmids and transfected into our immortalised cell lines generating an in vitro chronic wound reporter bioassay (Fig. 2).  We envisage, through future generation of this system, that such an in vitro bioassay will be utilized as a high-throughput pre-screening system to study the efficacy of agents to assist dysfunctional wound healing and in so doing replace a large number of animal experiments.

Figure 2: Chronic wound fibroblasts expressing two disease-specific reporter constructs

The Group also has an interest in the microbiology of these wounds and how this can directly and indirectly affect cellular behaviour.  Chronic venous leg ulcer (CVLU) wounds are colonised by a bacterial microflora, even when clinically non-infected and this is most often poly-microbial.  Our cultural analyses have documented that staphylococci, streptococci, enterococci and facultative Gram-negative bacilli are the bacterial groups most frequently recovered from CVLU, with almost 60% of CVLUs shown to harbour anaerobic bacteria such as the peptostreptococci.  Attempts have increasingly been made to investigate both disease association of specific bacterial groups and the relationship between microbial bio-burden and healing in chronic wounds.  To date, no consistent association with healing of these wounds has been demonstrated for any bacterial species.  In some studies it has been suggested that the number of colony forming units of Staphylococcus aureus per gram of tissue may represent a guide to non-healing.  However, this is not uniformly accepted and studies in other polymicrobial chronic infections suggest that specificity of micro-organism is more important than bacterial bio-burden.  We have shown that this is also the case for chronic wounds (Davies et al., 2007).  Furthermore, utilising 16S rRNA and PCR-sequencing as a tool for identifying difficult to culture micro-organisms we have revealed a significantly greater bacterial diversity within these wounds than that revealed by culture alone with the identification novel species of bacteria also (Davies et al., 2001; Hill et al., 2003; Davies et al., 2004).  We have gone on to show in our in vitro systems that these bacteria are detrimental to normal cellular wound healing function (proliferation, migration) through the production of secreted by-products such as short chain fatty acids (Wall et al., 2002; Stephens et al., 2003b).  We are now extending our microbiological investigations through the development and characterisation of wound biofilms within the lab (Fig. 3; Malic et al., 2007) and through an ongoing multi-centre, collaborative investigation centred on the development of a real time reporter dressing for wound infection.  A recent study has shown that chronic wound patients are routinely over-prescribed antibiotics (Howell-Jones et al., 2005) and we are now also investigating the role that this may play in increasing antimicrobial resistance of the wound microflora in this particular patient group. 

Preferential healing within the Oral cavity

Unlike skin wounds, oral mucosal wounds heal with little/no scar formation.  In vitro, we have demonstrated that oral mucosal fibroblasts (OMF) exhibit a distinct phenotype with increased extracellular matrix reorganisational ability, migration and experimental wound repopulation when compared to skin fibroblasts, which is linked to the differential production of proteases and growth factors (Stephens et al., 1996; al-Khateeb et al., 1997; Stephens et al., 2001a & b).  In relation to the lack of scarring of oral mucosal wounds we have also shown that OMF are resistant to transforming growth factor-β₁ driven differentiation to myofibroblasts (Meran et al., 2007; Meran et al., 2008).  We have also demonstrated recently that, compared to patient-matched skin fibroblasts (SF), OMF have a significantly increased lifespan (80-115 populations doublings compared to 40-65 populations doublings for SF) which is linked to the longer telomeres detected within these cells (Enoch et al., 2008). 

 Microarray analysis of OMF and SF has revealed that genes associated with resistance to oxidative stress are upregulated in OMF (Enoch et al., 2008) which in turn is linked to significantly lower ROS generation in OMF versus SF (Lohana et al., 2008).  Indeed, as these antioxidants are known to reduce telomere shortening rates, thereby extending replicative life-span, their increased expression in OMFs may be the reason for their extended proliferative lifespans in vitro and their superior wound healing abilities compared to SF.  Based on these findings, interventional studies, are ongoing in relation to our interest in chronic wounds, involving the retroviral infection of CWF to over-express antioxidants, to determine whether they can delay the onset of oxidative stress-induced, premature senescence in CWF.

We gratefully acknowledge funding for our research from Algipharma (Norway), Astratech (Sweden), Beiersdorf AG (Germany), Convatec Ltd, Diabetes UK, The Engineering and Physical Sciences Research Council, FMC Biopolymer (Norway), The Dr Hadwen Trust, Johnson & Johnson Wound Management, National Institutes of Health (USA), the National Centre for Replacement, Refinement and Reduction of Animals in Research, the Oral and Dental Research Trust, The Osteology Foundation (Switzerland), Research Into Ageing, Royal College of Surgeons (Lon), Royal College of Surgeons (Edin), The Royal Society, Veterans Affairs Medical Center (USA), The Wellcome Trust, Welsh Office for Research and Development for Health and Social Care

During ageing, there is a progressive onset of insulin resistance (Pagano et al., 1981), shown by aberrant regulation of glucose and protein metabolism. However, insulin resistance is not routinely treated unless diabetes develops. Data suggests that insulin resistance precedes sarcopenia, and may therefore play a fundamental role in its development (Rasmussen et al., 2006). There is mounting evidence in support of this hypothesis that sarcopenia may be a result of age-related insulin resistance (Fujita et al., 2007).

The molecular pathways that underlie the age-related development of insulin resistance in skeletal muscle are complex and incompletely understood. IR is associated with mitochondrial dysfunction that has been proposed to lead to accumulation of intramyocellular lipid and defective insulin signalling (Petersen et al., 2003).  Ageing is also associated with an increase in reactive oxygen species generation (ROS) and evidence suggests that this increase in ROS generation with ageing may lead to insulin resistance.  This suggestion that ROS and insulin resitance are intrinsically linked is supported by data showing that mice with a homozygous knockout for neuronal nitric oxide synthase (nNOS) have increased oxidative stress in muscle and also exhibit severe insulin resistance. Further data linking ROS generation and IR comes from studies of the anti-diabetic drug, Metformin that corrects IR. This compound has recently also been shown to stimulate AMP-activated kinases and enhance nitric oxide (NO) synthesis, which may reduce superoxide bioavailability and thus reduce oxidative stress. Once IR is established in muscle of old people and animals, this may potentially lead to a vicious cycle of increased blood glucose levels resulting in further oxidative stress, further ineffective increases in circulating insulin concentration and catabolic effects on the muscle (Leverve, 2003).

In ‘insulin sensitive' skeletal muscle the binding of insulin to its receptor activates 2 major signalling pathways; these pathways are the Ras-Raf-MEK-ERK pathway and the PI3K/AKT pathway. The first pathway does not appear to be involved in modifications to muscle fibre size (but is involved in fibre type composition) whilst the second pathway has major effects on muscle fibre hypertrophy and protein degradation (see Figure 1). Insulin stimulated phosphorylation of AKT increases protein synthesis via GSK and mTOR kinases. Furthermore, phosphorylation of AKT results in the downstream phosphorylation of the Forkhead box O (Foxo) class of transcription factors. In its phosphorylated state, FOXO remains in the cytosol, however dephosphorylated FOXO is capable of nuclear translocation where it triggers protein degradation through atrogenes such as MuRF1 and MAFbx (Sandri et al, 2004). Therefore we propose that ageing results in increased generation of ROS which results in the development of insulin resistance with impaired insulin signalling and consequently increased muscle protein degradation and decreased protein synthesis, resulting in sarcopenia. 

Figure 1. Schematic representation of the insulin signalling cascade demonstrating the effects of insulin on protein synthesis and degradation.

Dr Close is supported by a Fellowship award from Research into Ageing.

Over the past decade research into the molecular biology of ageing in model organisms revealed that "ageing" shares common denominators indicating conserved mechanisms over different taxa. Calorie or dietary restriction without malnutrition is the most powerful evidence for this observation, because it is the only non-genetic intervention which extends lifespan of organisms across taxa, i.e. yeast, worms, fruitfly, and rodents. Additionally, species- specific pathways have evolved (1).

Yeast is fortunate enough to have two lifespans. Chronological lifespan measures the time of survival in a non-dividing state. In contrast, the more widely studied replicative lifespan measures the number of replications a mother cell undergoes before it senesces, and is regarded as a model for mitotically active cells. Restricting glucose availability has been shown to extend both chronological and replicative lifespan (1), although most studies on calorie restriction in yeast have focused on replicative lifespan.

Why yeast cells age has not been unambiguously identified yet. There is evidence that accumulation of oxidatively damaged proteins in mother cells induce senescence over the replicative cycles, because the damaged proteins are selectively retained in the mother cell (2;3). In addition, a popular and long-standing theory suggests that ageing is caused by accumulation of extrachromosomal rDNA circles (ERCs), which arise during homologous recombination of rDNA-tandem repeats. Each of these recombined repeats is excised and forms a little circle, not unlike plasmids. Since they contain an origin of replication, they autonomously replicate during each cell division, and thus reach high numbers in old yeast cells; in fact their DNA content can outweigh the yeast genome. Again they are retained in mother cells, and it is thought that they titrate housekeeping factors away from the chromosomes, which leads to decreasingly strict maintenance of the genome (4).

Today, three pathways linking calorie restriction to lifespan extension have been suggested in yeast (Figure 1). The first two propose that Sir2, an NAD^+-dependent histone deacetylase which is required for gene silencing, is the effector via which calorie restriction exerts its beneficial effect on lifespan. Model I proposes that Sir2 senses shifts in nutrient-sources due to changes in the ratio of intracellular NAD⁺, which increases when yeast switch from fermentation (the rather inefficient energy-production when nutrients are abundant) to respiration, hence increasing the NAD⁺ to NADH-ratio. This leads to increased Sir2 activity and hence more silencing, thus increasing genome stability and reducing the ERC-formation, which slows ageing and thus extends lifespan (5). Model II explains the increase in Sir2-activity via the upregulation of the NAD⁺-salvage pathway in the nucleus which leads to a decrease in nicotinamide, an inhibitor of Sir2. This again results in Sir2-upregulation and feeds in the same pathway as does model I (6).

Model I and II are widely accepted as mechanisms for lifespan extension. Model III however omits Sir2 completely, and there is evidence that the Tor-pathway, and Sch9, the yeast PKB/AKT- homologue coordinate responses to calorie restriction and control lifespan extension. However, the further downstream mechanisms are still a black box, and it is not clear whether these pathways also modulate events at the rDNA (7;8).

In order to assess changes in gene silencing in response to calorie restriction, we are using a simple, but in its read-out very sensitive silencing assay. Because Sir2 is the only histone deacetylase required for silencing in the telomeres, the mating type locus and the rDNA, we chose to first assess its activity in the telomeres. Therefore, we worked with a reporter strain which has the marker gene URA3 inserted in the subtelomeric region. URA3 codes for an enzyme which is involved in the pyrimidine biosynthetic pathway. It converts 5'-Fluororotic acid into 5'-Fluorouracil, which is highly toxic for cells. In wildtype cells, where Sir2 is expressed and active, this gene is silenced and cells will grow on FOA-supplemented media. In a Sir2-deletion strain however, silencing is abolished, therefore URA3 is expressed, and the cells die on FOA-supplemented media. This simple assay allows for a qualitative and quantitative analysis of influences which directly act on Sir2, such as calorie restriction. (Fig 2.)

Cells are then grown on normal and FOA-containing media which are supplemented with the following range of glucose-concentrations: 0.05% and 0.5% for severe and moderate calorie restriction, and 20% for high external osmolarity, which was also shown to extend lifespan via Sir2 upregulation (9), and 2% glucose, which is the standard growth condition.

Additionally to the parent strain, we constructed a set of strains with gene deletions which are all reportedly involved in modulation of lifespan. The deletion of the replication fork barrier protein Fob1 is thought to extend lifespan via the reduction of ERCs. The deletion of the gene for hexokinase isoenzyme 2 HXK2 is generally regarded as the mimic for calorie restriction and also extends lifespan. The deletion of Sir2 leads to reduction in lifespan.

Our data show that Sir2-activity does not change in response to calorie restriction. Therefore, lifespan extension by calorie restriction in yeast is not due to an increase in Sir2 activation. However, we found increased gene silencing in the fob1Δ and hxk2Δ strains. The effect of calorie restriction in the hxk2Δ strain is an important finding, because it indicates that this deletion has additional effects on an organism and it does not exactly mimic calorie restriction. The effect in fob1Δ could be due to a redistribution of Sir2 away from the rDNA, where it is normally recruited to by Fob1.

In order to exclude locus-specific effects at the telomeres, and to assess whether model III provides better evidence for lifespan extension by calorie restriction, we are currently performing a series of silencing assays using a reporter strain with the marker inserted in the rDNA locus. Preliminary data that calorie restriction does not increase silencing at rDNA either, implying that the Sir2-independent pathway of model III may be correct. Future experiments are aimed at understanding the molecular mechanisms which lead from calorie restriction to lifespan extension.

Figure 1. Three proposed models connecting calorie restriction with lifespan extension in yeast. Model I and II suggest an upregulation of the histone deacetylase Sir2 via two different pathways. Model III has not been well defined mechanistically; however, the TOR and SCH9 kinase pathways have been suggested to be involved.

Figure 2. Sir2 activity regulates silencing of the URA3 reporter gene. In the wildtype strain, Sir2 silences the reporter gene introduced in the subtelomeric region and the cells survive on FOA-containing medium. In the Sir2 deletion strain, URA3 is expressed, and cells die on FOA.

Several lines of evidence suggest a link between genome integrity and ageing, but perhaps the most compelling is the premature ageing seen in mammals that have defects in DNA-repair processes.  Typically, the mechanism linking DNA damage to ageing is postulated to be mutation accumulation or increased cell death.  However, we pose the question: if DNA repair was 100% efficient and there is no opportunity for mutations to accumulate, would the very presence of ongoing repair processes have an effect on the physiology?

To investigate this idea our laboratory has become increasingly interested in the premature ageing Werner syndrome (WS). Clinically, WS individuals prematurely show many features of normal ageing including, greying hair, aged skin, and cataracts, and show pre-disposition to age-related diseases such as type II diabetes, atherosclerosis, and osteoporosis¹.  Additionally, there is an elevated susceptibility to rare mesenchymal tumours.  WS is caused by loss of the RecQ helicase WRNp, resulting in defects in DNA repair and recombination² that lead to chromosomal rearrangements and increased replication fork stalling³.

The molecular mechanism of in vivo ageing in WS appears to be associated with accelerated cell ageing. WS cells have a shortened in vitro lifespan and resemble normal senescent cells in that they are enlarged, flattened and granular, even at a young age, and have prominent F-actin stress fibres reminiscent of cells undergoing stress-induced premature senescence (SIPS) ⁴.  Recent studies suggest that the shortened lifespan reflects a synergy between telomere-driven senescence and a novel telomere independent senescence mechanism that involves the p38^MAPK stress-signalling pathway⁴.  We hypothesise that increased replication fork stalling in WS cells is sensed as replication stress that triggers p38 activation leading to SIPS.  Treatment with the p38 inhibitor, SB203580, rescues the senescent-like morphology of WS cells and increases the lifespan and growth rate to within the normal range, i.e., effectively preventing premature cell ageing in WS⁴.  This suggests that p38 activation contributes to the abnormal cellular phenotype in WS and, furthermore, that some of the clinical phenotypes, such as the elevated rates of inflammatory disease, including type II diabetes, atherosclerosis, and osteoporosis seen in WS, may in part be due to chronic activation of p38. WS thus provides a powerful system to dissect the interplay between senescence, genome instability and cancer.

However, the question remains as to whether this phenomenon is simply a unique "private" mechanism at work in WS, or is a general ability of cells to respond to physiological levels of cell intrinsic genome instability.  To address this, further study is underway to investigate whether a similar response is seen in genome instability syndromes such as Rothmund-Thomson, Bloom, Hutchinson-Gilford, Nijmegen-breakage (NBS1) and Cockayne (CSA), amongst others.  Initial studies focus on determining whether primary fibroblasts show any of the changes in cell behaviour that are seen in WS, e.g., changes in growth rate, cell morphology, production of stress fibres and p38 activation.

Morphologically, fibroblasts from other genome instability syndromes show similarities to WS in that they are enlarged and granular displaying distinct stress fibres.  Of particular note is the fenestrated appearance seen in many of the cells, most prominently in NBS1 and CSA.  Treatment with p38 inhibitors, such as SB203580, BIRB796 and VX745, show varying effects in the different syndromes, e.g. they dramatically rescue the senescent-like and stress fibre phenotypes of NBS1 fibroblasts, but have little effect on Bloom syndrome fibroblasts.

To date, evidence suggests that fibroblasts from all of the syndromes show aberrant physiology, suggesting that that they are undergoing some form of stress, perhaps in response to ongoing genome instability. However, preliminary studies with p38 inhibitors suggest that, either p38 is not active in all cell strains, or indeed if it is, it does not always contribute to their phenotype. Therefore, it seems that p38^MAPK upregulation is specific to WS, and that while genome instability per se might trigger a stress-signalling cascade, this may not exclusively involve p38.  

Figure legend

Effect of BIRB796 on RTS fibroblasts, shown by phase contrast.   Untreated cells (left) and BIRB796 treated cells (right).

BCA activities

The British Council for Ageing (BCA) aims to promote understanding between the different disciplines involved in the study of ageing and older people. In its first 6 months of operation it has carried out activities in the area of capacity building, training, awareness raising and policy making. Some examples of key activities are:

• 2 workshops for 2007, run jointly with SPARC (Strategic Promotion of Ageing Research Capacity, funded by BBSRC and EPSRC). The first workshop held at Birmingham University in February had 70 attendees and concerned funding available in FP7 for Ageing Research. As a result at least 6 applications are being written for submission to FP7 in 2007. The second workshop, organised by Professor Chris Phillipson (BSG), will take place on May 24th at Keele University and concerns teaching of Ageing in the undergraduate curriculum. It will have speakers from all three societies and aims to produce a cross-disciplinary curriculum for medical students and identify participants willing to work together to produce a lecture notes series and/or short textbook suitable for undergraduate courses in the area of social gerontology and biogerontology.

• Professor Janet Lord (Chair of BSRA) represented the BCA at a SPARK workshop organised by the Funders Forum for Research on Ageing and Older People (FFRAOP) and Unilever on The Future of Ageing Research in the UK. A document outlining the conclusions of this workshop has been published recently (O.H.Franco et al. (2007) Ten commandments for the future of ageing research in the UK: a vision for action. BMC Geriatrics 7:10. Available at www.biomedcentral.com/bmcgeriatr/).

• Professor Lord will also represent BCA and BSRA at a second SPARK workshop on The Healthy Ageing Phenotype organised by the MRC and Unilever on May 24^th and 25^th in Amsterdam.

• The BCA have been invited to attend FFRAOP as observers, which will give the opportunity to contribute to discussions on the future policy on Ageing Research in the UK.

• BCA advised upon the content of the "Ageing" section of a public display at the Centre for Life in Newcastle. The display hopes to stimulate the general public to think about the Future with respect to the older adult and what current research might suggest as possible significant changes to how we will live our lives as older people in the future. The exhibition opens on May 24^th.

• BCA was invited to, and had a stand at, the European FP6 funded workshop entitled AGEACTION: Changing Expectations of Life, at The SAGE conference centre, Newcastle on Tyne, April 23^rd 2007. The stand was visited by many of the 450 delegates.

• Professor Lord was invited to a roundtable briefing with the Minister for Science Malcolm Wicks at The Royal Society on April 19^th 2007. The briefing was to discuss current progress in Biology of Ageing Research, but issues relating to geriatric medicine and social gerontology were also discussed. The briefing was attended by the science correspondents of several national newspapers (The Guardian, The Times, The Daily Telegraph, Daily Mirror and Daily Mail).

Skeletal muscle strength declines significantly during ageing and muscle of aged mammals also is more susceptible to exercise-induced damage and has an impaired ability to fully recover from damage. This leads to instability, an increased risk of falls and an inability to perform everyday tasks, reducing the quality of life of older individuals. The mechanisms underlying these age-related functional deficits are unclear. Muscles of young individuals can adapt following exercise by the increased production of heat shock proteins (HSPs) which protect the muscle during potentially damaging exercise. In contrast the induction of HSPs following exercise is attenuated in muscles of old individuals (Vasilaki et al. 2003). Previous studies using transgenic mice demonstrated a direct link between failure to produce HSPs and functional deficits in muscles of old mice whereby overexpression of HSP70 in skeletal muscle throughout life provided some protection against the development of age-related functional deficits (McArdle et al. 2004).

This study hypothesised that the development of age-related muscle weakness is due to the failure of adaptation, particularly in the HSP response following contractile activity. Maintenance of the HSP content of old muscles at rest and following contractile activity may result in significant improvement in muscle function. The study aimed to examine physiological (treadmill training) and pharmacological interventions that may preserve the ability of muscles of old mice to induce HSPs in skeletal muscle and to assess the effect of these interventions on the susceptibility and recovery of EDL muscles from damaging exercise.

Data demonstrated that 8 weeks of training with protocol of 3 sessions per week for 15 minutes per session at 15m/min was sufficient to cause adaptations in quadriceps muscles of adult mice including changes in redox homeostasis and increases in the HSP70 and HSC70 content. Once this protocol was established, adult (12 month old) mice were trained for 12 months and old (22 months) mice were trained for 10 weeks. To assess the ability of muscles to produce HSPs following exercise, hindlimb muscles of trained and control mice were subject to a 15 minute protocol of non-damaging isometric contractions. Data demonstrated that treadmill training prevented changes in resting levels of HSP70 protein and mRNA and preserved the ability of muscles of old mice to produce mRNA for HSP70 to the same level as adult mice following the non-damaging exercise. To assess the functional effect of this intervention, the maximum tetanic force of the EDL muscle was determined. The EDL muscles were subjected to 450 damaging lengthening contractions. The ability of muscles of these mice to generate force was assessed at 3 hrs and 28 days following the contractions to determine the susceptibility to and recovery from damage. Data demonstrated that treadmill training did not correct the age-related loss of force, protect against a severe protocol of damaging lengthening contractions or enhance recovery.

Pharmacological investigations demonstrated that treatment with the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG) significantly increased the HSP70 content of skeletal muscle cells both in vitro and in vivo. HSP70 remained elevated in muscle of adult mice for up to 6 days following a single dose. Adult and old mice were then treated for 4 weeks with 17AAG. Data demonstrated that 17AAG significantly increased HSP70 in muscle of adult and old mice via activation of the transcription factor HSF1. This did not affect markers of oxidative stress or prevent the loss of force in muscles of old mice.

In summary, data have suggested that short- or long-term treadmill training or treatment with 17-AAG resulted in some maintenance of the ability of muscles to produce HSPs. However, the lack of protection against the development of functional defects suggests that this was insufficient to be of significant benefit to muscles of old mice.

This work was funded by Research into Ageing.

My PhD thesis involves the experimental analysis of the mechanisms of telomere loss during the onset of replicative senescence, the potential nature of the telomere signal that triggers replicative senescence, and the possible contribution of telomere dynamics to aging and chromosome instability. Human telomeres are composed of the DNA sequence TTAGGG reiterated into arrays of upto 20kb, which together with associated proteins cap the ends of human chromosomes. The loss of telomeric sequences with ongoing cell division due to the end replication problem, ultimately results in the loss of telomeric function and the triggering of replicative senescence in many human cell types. Telomere erosion can therefore impose a limit on the replicative potential of human cell. This raises the possibility of a telomere-dependant accumulation of senescent cells in human tissue, which may underline age-related tissue deterioration, disease and cancer predisposition.

I have been investigating telomere dynamics using the Single Telomere Length Analysis (STELA), a high resolution technique that determines telomere length from single DNA molecules at specific chromosome ends. I have extended the orginal observations at the XpYp telomere by extending STELA analysis in include the telomeres of 17p, 11q, 12q, and 2p. Analysis of fibroblast populations passaged to senescence revealed two mechanisms that result in telomere loss; gradual erosion as a consequence of end-replication losses, and large-scale length changes termed human Telomere Rapid Deletion (hTRD). With the exception of 17p, the telomere dynamics were conserved amongst the telomeres analysed. However, the 17p telomere was more stable with a striking paucity of hTRD and displayed a trend towards being the shortest telomere, indeed this end exhibited the shortest recorded telomere length distribution (300bp) in senescent cells.. The mutational mechanisms that underlie hTRD is unclear. However it appears likely from genetic data in human cells ans observations in yeat that hTRD may involved intrachromatidal processes such as recombination with the telomere loop sgtructure, oxidative damage and sister chromatid exchange. The mechanisms underlying these mutational events are currently under investigation.

The ectopic over expression by the retrovirall transduction of the telomerase catalytic subunit (hTERT), resulted in the homogenisation of both allelic telomere length distributions and chromosome specific length distributions. However analysis of cancer derived keratinocytes expressing physiological levels of hTERT, revealed the maintenance of allelic distributions and chromosome specific lengths, where 17p was again the shortest analysed telomere. This phenomenon has also been shown in the male germ line, where 17p was consistently the shorter telomere, compared to XpYp. Such results suggest the presence of cis acting determinates of chromosome and indeed allele specific telomere lengths, that operate in the presence of telomerase.

Winning the best poster prize at the BSRA meeting in Oxford was an immense privilege, especially due to the quality of the other entrants. Winning this prize has boosted my confidence concerning presenting my work for public viewing, and has reassured me that my work is of reasonable quality. Winning the prize has also increased my incentive to work harder, and to improve the standard of presenting my work at other scientific meetings. I would like to thank the BSRA for the travel grant, and the thought provoking well organised 2006 meeting. I would also like to thank my amazing PhD supervisor, Duncan Baird for all his support and guidance.