Mitotic cells

Senescent phenotype and biological impact

Senescent cells tend to adopt an extracellular matrix (ECM) degrading, proinflammatory phenotype (West et al, 1989; Kletsas et al, 2004). Senescent cells usually up-regulate matrix metalloproteinases (MMPs), enzymes capable of degrading proteins such as collagen and elastin which make up the extracellular matrix. Since the extracellular matrix (ECM) is important for providing support and anchorage for cells, separating different tissues and regulating intercellular communication, its degradation by MMPs is likely to impact all areas of ECM function. MMP activity is normally inhibited by TIMPs (tissue inhibitor of metalloproteinases), but research suggests that that these inhibitors themselves are down-regulated at senescence, thereby further contributing to matrix degradation (Hornebeck, 2003).

Senescent cells also secrete many cytokines which due to their diverse function could have multiple consequences on the ageing of tissues. These secreted proteins may not just impact on local tissue but also tissues found throughout the organism. The presence of cytokines can alter cell functions by up-regulating or down-regulating several genes and their transcription factors, resulting in the production of other cytokines and an increase in the number of surface receptors for other molecules (Gallin and Snyderman, 1999). The ability of cytokines to reach many tissues and have such diverse consequences on cell function suggests that only a small fraction of senescent cells may need to be present for there to be any significant impact on tissue impairment or disease development.

As discussed in post-mitotic ageing, the accumulation of senescent cells in some tissues is likely to reduce the number of cells which can provide support and protection to post-mitotic cells. Therefore, the appearance of senescence cells may have a direct impact on the impairment of post-mitotic tissues.

Some of the changes observed during cellular senescence are also likely to be cell type specific. Different cell types are going to have different transcriptional profiles since their functions are different and these differences may result in tissue specific impairment. For example, in senescent vascular endothelial cells, nitric oxide synthase (eNOS) activity has been found to be decreased (Matsushita et al, 2001; Minamino et al, 2002). Since nitric oxide (NO) is important in regulating vascular function, a decline in its production may have detrimental consequences. A reduction in NO production by eNOS for example has been suggested to be a significant risk factor for cardiovascular disease (Cannon, 1998). This decline in eNOS activity at senescence appears to be specific to vascular endothelial cells. Even if eNOS is produced by other cells types and a similar decline with age is observed, the consequence of such changes is going to be different, if any at all. This is due to alterations in specific structure-function relationships.
Overall, senescent cells within tissues are thought to contribute to the ageing process by:

1) Altering the behaviour of neighbouring growth-competent mitotic cells.
2) Degradation of structural components such as the extracellular matrix.
3) Reducing the pool of growth-competent mitotic cells.
4) Cellular dysfunction: inability to function properly.

Mitotic cells

Mitotic tissues consist of cells which have the ability to divide when stimulated. Most mitotic cells (i.e. fibroblasts, endothelial cells, smooth muscle cells, glial cells, astrocytes etc) within tissues are found in a reversible growth arrest known as quiescence. These cells remain quiescent until stimulated to proliferate, usually for the purpose of cellular replacement. How often these cells proliferate is dependent upon how frequent cells become damaged or lost, and this may be connected to the function of the tissues in which they reside. For example, fibroblasts exposed to environmental UV radiation or endothelial cell in blood vessels exposed to high turbulence in blood flow may be more likely to proliferate to replace cell loss than less damage prone tissues.

Overview of cellular senescence

The predominant ageing mechanism of mitotic tissue is thought to be due to the gradual accumulation of senescent cells. Senescent cells have undergone an irreversible cell cycle arrest, and display a radically altered phenotype: genetically, morphologically and behaviourally distinct from its growth-competent counterparts. The accumulation of these dysfunctional cells is thought to result in a gradual decline in tissue function and the manifestation of age-related disease.

One of the mechanisms for triggering cellular senescence is thought to be due to the presence of a critically short telomere. Telomeres are regions of highly repetitive DNA at the end of linear chromosomes, which are bound by a number of proteins which are thought to protect the telomere from being processed as DNA double strand-breaks. Every time a cell divides the telomeres become progressively shorter due to the inability to replicate DNA at the ends of chromosomes (Joosten et al, 2003). This would eventually result in the appearance of a short telomere, which can no longer be protected by telomeric proteins. This may lead to the exposure of DNA ends, resulting in a DNA damage response. This response is thought to cause the cell to enter what is known as “replicative senescence”.

If replicative senescence is essentially the result of the exposure of DNA ends, oxidative stress causing DNA damage such as DNA double strand breaks may also be a mechanism for triggering cellular senescence (Von Zglinicki et al, 1998). This idea is supported by data demonstrating that the signalling pathways connecting telomere shortening and cellular senescence is similar to the one that is activated by DNA damage (Von Zglinicki et al, 2005). The mechanism (ROS or replicative senescence) thought to be predominantly responsible for cellular senescence in tissues is currently unknown. The appearance of senescent cells within mitotic tissues is going to be dependent upon three main factors:

1) The rate of cell turnover.
2) The replicative lifespan of the cells.
3) The survival time of senescent cells in vivo.

The rate of cell turnover is dependent upon the rate of cell loss. Cells divide to replace lost cells. Tissues with a high cell turnover rate are much more likely to exhaust their replicative capacity and consequently increasing the likelihood of cells entering senescence. Different cells have different replicative capacities, some cell types may be able to divide a maximum of 100 cumulative population doublings (cPD) (Poiley et al, 1978) before entering senescence while other cell types a maximum of only 20-30 (cPD) (Kalashnik et al, 2000). Therefore, if the rate of cell turnover for all cell types was constant, some tissues are still more likely to enter senescence than others. There are at present no studies that have looked at the survival time of senescent cells in vivo. However, it has been demonstrated that senescent fibroblasts can be maintained in culture medium for years. If the survival time of senescent cells was short, then the loss of such cells would result in further cell turnover and further reduction in the proliferative capacity of the tissue. Research has suggested that senescent cells tend to be more resistant to apoptosis (and thus more likely to persist in tissues), at least in fibroblasts where most of the research has been conducted (Wang et al, 1994, 2004, Marcotte et al, 2004, Hampel and Wagner, 2005). However, experimental evidence measuring the fraction of apoptotic human vascular endothelial cells also demonstrated no difference between the apoptotic potential of senescent cells compared with their mitotic counterparts (Kalashnik et al, 2000). If there is no increase in the apoptotic potential of senescent cells, it is likely that these cells persist in tissues for long periods of time, thus causing damage.

Post-mitotic tissue

Damage to proteins

Damage from ROS is also thought to have an age-related impact on proteins. Such damage is thought to result in amino acid modifications, fragmentation of peptide chains, altered electrical charge and protein aggregation (Davies, 1987). Since the structure of proteins is pivotal for performing its functions then any structural changes would therefore result in an impairment of function. As discussed previously, the turn-over rate of proteins is an important factor in determining whether any protein damage is going to have a significant affect. Since the majority of intra-cellular proteins have high turn-over rates, damage proteins are not going to persist long enough to cause any problems. Therefore, long-lived proteins which may be affected by AGE formation may also be affected by oxidative damage. Examination of extracellular tissues in the lens, skin collagen and articular cartilage (low turnover proteins) of humans ranging in age from infancy to 80 years showed an increase in oxidative markers with age (Linton et al, 2001). The same study also looked at intracellular proteins with high turnover rates and found no evidence to suggest that intracellular proteins accumulate oxidative damage with age.

Damage to Lipids

Lipids which make up the membranes of cells are also potential targets for ROS which may consequently result in biologically significant alterations to membrane proteins. ROS are thought to attack membrane phospholipids and act on unsaturated fatty acids to produce lipid peroxidation. The consequent of this may be alterations in membrane fluidity, increased permeability and loss of membrane integrity. Experimental evidence suggests that altered membrane fluidity might affect permeability, transport systems, receptor functions or enzyme activities (Stark, 2005). The functionality of proteins in the membrane is critically dependant on membrane fluidity, especially when proteins have to collide with other molecules to exert their effects (such as G-proteins). This is seen in many receptor mediated pathways. For example, cardiac membranes from rats with cirrhotic cardiomyopathy are rigid and associated with diminished cAMP production. When the fluidity of these membranes are restored to control values cAMP production was significantly increased (Ma et al, 1997).

Biological impact of ROS

The above discussion on ageing of post-mitotic cells reviews the mechanisms thought to result in an overall increase in cellular damage by ROS, but little evidence is provided for the age-related biological consequence of such damage. If accumulative damage from ROS is an ageing mechanism of post-mitotic tissue, then such damage may result in three possible outcomes:

1) Post-mitotic cells become damaged and are subsequently removed. Post-mitotic-cells cannot be replaced, thereby resulting in a decrease in overall cell number and a decrease in tissue function.

2) Damage to post-mitotic cells results in an impairment of cellular function but such damage is not extensive enough for the removal of cells and so they persist.

3) Cells are removed due to damage and are subsequently replaced (i.e. by mitotic cells). However, the ageing mechanism in this case may therefore be a decline in the capacity to replace cells due to an ageing mechanism specific for mitotic tissues which will be discussed later. This may result in an observation similar to point 1).

The literature was reviewed to identify whether these three possible outcomes are present in ageing of post-mitotic tissues. One study observed a substantial increase in the amount of DNA single strand breaks in hippocampal pyramidal and granule cells as well as cerebellar granule cells but not in cerebellar Purkinje cells in the brain of ageing rats (Rutten et al, 2007). However, a reverse pattern was found for age-related reductions in total numbers of neurons. Cerebellar Purkinje cells were found to be significantly reduced during ageing (point 1 above) whereas the total number of hippocampal pyramidal and granule cells as well as cerebellar granule cells were not (points 2 or 3 above). This may seem as a confusing result since those cells which have undergone the most DNA damage may be expected to decline in numbers. This result may be explained if there are processes in place that replace damaged pyramidal and granule cells but do not, or cannot, replace damaged Purkinje cells. Cells that may be more prone to damage may have a higher potential capacity to be replaced than cells that are less prone to damage. It is also possible that the damage inflicted on hippocampal pyramidal and granule cells is not severe enough to warrant their removal. These findings suggest that if ROS does cause detrimental damage leading to an ageing phenotype, such damage is not tissue specific damage, but rather, cell type specific. All post-mitotic cells cannot therefore be treated equally.

A decline in cell numbers may not be related to damage from ROS but instead due to the loss or dysfunction of mitotic cells such as glial cells and astrocytes which are known to provide support and protection for neurons. Discussed later in more detail in ageing of mitotic tissue, mitotic cells can undergo an irreversible cell cycle growth-arrest known as replicative senescence. Senescent cells display a radically altered phenotype with potentially detrimental consequences on other cells if they accumulate in tissue. For example, a recent study has demonstrated that alterations in astrocyte function with ageing may not only affect its neuroprotective capacity, but may also contribute to neuronal injury in age-related neurodegenerative processes (Pertusa et al, 2007). Also, microglial, cells involved in immunological surveillance and neuroprotection have been shown to be subject to replicative senescence and it has been suggested that the dysfunction of these cells may contribute to the development of neurodegenerative disease by diminishing glial neuroprotection (Streit, 2006).

Myocytes, also known as muscle fibres are post-mitotic cells found in skeletal, smooth and cardiac muscle. Studies have shown that ageing muscle results in a decline in myocyte numbers, signs of atrophy and increased susceptibility to contraction-induced injury (Alnaqeeb and Goldspink 1986, Musaro and Rosenthal, 1999, McArdle et al 2002,). When myocytes become damaged and need to be repaired, mitotic satellite cells are able to differentiate and fuse to augment existing muscle fibres and to form new fibres. Decline in myocyte numbers and increases in damage with age may therefore, at least in part, be due to a reduction in number or the impairment of satellite cells (point 3). Such a reduction in satellite cell numbers would result in a decrease in cellular maintenance and an increase in cellular damage, possibly leading to the removal of the cll altogether. One study has found that the abundance of satellite cells does appear to decline with age, however, the myogenic potential of these cells does not diminish with age (Shefer et al, 2006).

Post-mitotic tissue

Failure to repair oxidative damage

The third factor, which is thought to result in an increase in damage from ROS, is the functional decline in the ability to repair DNA damage. The common repair mechanisms include; base excision repair (BER), nucleotide excision repair (NER), mismatch repair (MMR) homologous recombination (HR) and non-homologous end joining (NHEJ). However, repair to DNA normally take place during DNA replication when cells are dividing, but post-mitotic cells do not divide. The main concern in this instance is not whether there is a decrease in the ability of cells to repair DNA with age, but instead whether post-mitotic cells can themselves repair DNA, if at all they need to.
DNA repair in post-mitotic cells

To determine whether DNA repair occurs in post-mitotic cells, a number of studies have specifically concentrated on neural tissue and have found that these tissues do have the capability to repair DNA damage. Most oxidative damage is repaired by BER pathway, which is initiated by specialised DNA glycosylases. Glycosylases are involved in the removal of damaged DNA in the first step of the BER pathway. Newly discovered glycosylases (NEIL1/2) have been found to remove DNA damage from bubble structured DNA, suggesting that NEILs favour repair of transcribed or replicated DNA (Englander et al, 2006). This suggests that DNA replication may not be necessary for the repair of DNA. Measurements of expression and activity of BER during the neuronal transition from proliferative to postmitotic state demonstrated a decline in BER expression and activity, but expression of NEIL1 and NEIL2 glycosylases increased (Englander et al, 2006). The removal of damaged DNA from bubble structured DNA was found to be retained in post-mitotic neurons. This suggests a role for NEIL glycosylases in maintaining the integrity of transcribed DNA in post-mitotic cells.

Other repair mechanisms have also been shown to be present in post-mitotic neurons. Nuclear extracts from human brain neurons have demonstrated that the adult mammalian brain has the ability to carry out MMR (Brooks et al, 1996). Another study also showed the presence of DNA repair activity in neurons and found that this activity correlates with an increase expression of both HR and NHEJ DNA repair factors (Merlo et al, 2005). In regard to NER activity, it was found that neurons do exhibit NER activity and this activity is lower than that found in fibroblasts (Yamamoto et al, 2006). These studies therefore suggest that DNA repair mechanisms do occur in post-mitotic cells.

Since post-mitotic cells have the ability to repair DNA without replication it has been suggested that post-mitotic cells may only repair their expressed genes with little concern for removing damage from most of the genome (Nouspikel and Hanawalt, 2002, 2003). This accumulated damage in silent genes of post-mitotic cells may eventually result in triggering cell death, especially if such cells express these genes in an attempt to resume DNA replication.

Repair mechanisms

Very few studies have sought to determine whether DNA repair mechanisms change with age (Engels et al, 2007). One study however measured NHEJ activity to repair DNA from double strand breaks in extracts prepared from isolated neurons from neonatal, young adult and old rat cerebral cortex (Vyjayanti et al, 2006, Rao, 2007). It was shown that cohesive end-joining activity decreases significantly with age, but blunt and non-matching ends were poorly repaired at all ages. Interestingly, another study has shown that repair of DNA double strand breaks by the NHEJ pathway is deficient in Alzheimer’s disease (AD) (Shackelford, 2006). Another study looked at base excision repair using brain and liver nuclear extracts prepared from mice of various ages (Intano et al, 2003). An 85% decline in repair activity was observed in brain nuclear extracts and a 50% decrease in liver nuclear extracts prepared from old mice compared with 6-day old mice. DNA MMR system has also been investigated in T cells at various stages of the T cell lifespan (Annett et al, 2005). No clear pattern in DNA mismatch frequency with increasing culture age was observed, but the ability to repair induced DNA mismatches revealed an age-related decline in the efficiency of the MMR system.

If DNA damage or mutation frequencies increase with age, the impact, if any, that these changes have on the ageing phenotype is currently unknown. However, genetic disorders in which DNA repair mechanisms are defective may provide some answers. One such disorder known as xeroderma pigmentosum (XP), show the consequences of inherited defects in NER, which include UV hypersensitivity, cancer predisposition and accelerated ageing of skin, lips, tongue and mouth (Lehman, 2003). This accelerated ageing appears to be due to increase damaged as a result of excess environmental factors such as sun damage. Since accelerated ageing is not observed in less exposed tissues, it can be assumed that the lack of NER activity has little or no impact on ageing, and this may partly be due to protection from antioxidant enzymes.

Post-mitotic tissue

Decrease in antioxidant defences
Age-related decreases in antioxidant defences are the second factor thought to leave tissues vulnerable to damage by ROS. As such, the expression and activity of the major antioxidant enzymes, glutathione peroxidise, glutathione reductase, superoxide dismutase (SOD) and catalase have been investigated in depth. For example, one group assayed these enzymes in both the epidermis and dermis of young and old hairless mice (Lopez-Torres et al, 1994). Catalase, SOD and glutathione reductase were shown to have similar activity levels in young and old rats with glutathione peroxidise activity decreasing in old mice. It was concluded that skin ageing is not accelerated with age due to a general decrease in the antioxidant capacity of the tissue. A similar result was observed with the measurement of glutathione levels in human plasma from age-related macular degeneration (ARMD) patients, non-ARMD diabetic patients, aged non-ARMD and non-diabetic individuals and young individual without ARMD and diabetes (Samiec et al, 1997). No difference in glutathione levels was observed in aged or ARMD individuals but a decrease in glutathione was found to be associated with diabetes. Another study specifically concentrated on expression and activity of two SOD isozymes (Mn SOD and CuZn SOD) in three different skeletal muscle fiber types of young and old rats (Hollander et al, 2000). This study found that despite a decrease in mRNA expression in ageing muscle, an increase in enzyme activity was observed. Therefore, it was suggested that mRNA levels is not a determinant of SOD production, but due to post-transcriptional and/or post-translational mechanisms. Increases in enzyme activity have also been reported in a study which sought to characterise age-related changes in glutathione peroxidise, SOD and catalase in the rat aorta of young middle aged and old animals (Demaree et al, 1999). Glutathione peroxidise activity was found to increase with age, whereas SOD decreased in middle age before gradually increasing in old rats. Catalase activity was found to decrease significantly between young and old rats.

These increases in antioxidant enzymes with age may be an attempt to combat increasing attacks from elevated levels of ROS. If cellular damage is occurring despite the presence of antioxidant enzymes, it may be because these enzymes are not 100% efficient resulting in a gradual accumulation of damage. Over-expression of antioxidant enzymes have been shown to extend lifespan. For example, the over-expression of catalase in transgenic mice extended lifespan on average by 5 months (Schriner et al, 2005). These mice also showed a delay in cardiac pathology, cataract development and a reduction in oxidative damage and mitochondrial deletions. Over-expression of catalase and SOD has also been shown to impede the development of atherosclerosis in ApoE-/- mice (Yang et al, 2004).

Interestingly, a number of groups which generated mice lacking a particular antioxidant enzyme, found that these mice generally develop normally with no affect on lifespan. Mice lacking catalase were found to develop normally and showed no difference in hyperoxia-induced lung damage or increase susceptibility to oxidative stress in the lenses compared with wild type mice (Ho et al, 2004). Similarly, mice lacking glutathione peroxidise showed no difference with control mice in regard to longevity, vitality, weight, lens biochemistry or morphology (Spector et al, 2001). The absence of extracellular SOD in mice also showed no effect on lifespan, but these mice were more sensitive to hyperoxia than control mice (Carlsson et al, 1995 and Sentman et al, 2006). There are three possible explanations for why there appears to be no difference in lifespan of antioxidant enzyme lacking mice:

1) One antioxidant enzyme counteracts for the loss of another.

2) Repair mechanisms are sufficient to cope with any excess damage resulting from loss of an antioxidant enzyme.

3) Oxidative damage is not responsible for an ageing phenotype.

Post-mitotic tissue

The predominant ageing mechanism of post-mitotic cells (i.e. neurons, myoblasts and osteocytes) is often thought to be due to the accumulation of damage to DNA, protein and lipids caused by reactive oxygen species (ROS). Since these cells do not divide in tissues and can potentially persist for a lifetime, they are more likely to accumulate damage which may subsequently result in an age-related phenotype. Also, since mitotic cells are mostly found in a non-dividing state in vivo known as quiescence, with very little cell turnover, they could also in some regard be treated similar to post-mitotic cells.

ROS are thought to cause accumulative damage to DNA, protein and lipids, but how and if such damage results in an ageing phenotype still remains unclear. Damage from ROS is thought to be imposed on tissues as we age due to three possible factors:

1) An increase in ROS production.
2) A decrease in antioxidant defences.
3) A failure to repair oxidative damage.

Increase in ROS

Increases in ROS production is thought to be the result of mitochondrial dysfunction which leads to an increase in electrons leaking out of the respiratory chain within the mitochondria as we age (Wei et al, 2001). This increase in ROS is thought to increase damage to genomic DNA, mitochondrial DNA, proteins and lipids. Despite this theory, there appears to be little evidence to suggest that the levels of oxidative stress increases with age. One study however investigated age-induced ROS generation in healthy subjects ranging in age from 20-80 years quantifying ROS production using a chemiluminescence assay (Chaves et al, 2002). Results demonstrated a significant increase of ROS production from 40 years of age suggesting ROS does increase with age. However, little is known about the source of ROS. As mentioned, respiratory chain dysfunction leading to electron leakage is one suggestion.

With this in mind, a mutator mouse was created to investigate whether the mutations which were introduced in the mitochondrial resulted in mutant mitochondrial proteins that are defective in coupling of oxygen metabolism with ATP causing increased ROS production (Trifunovic et al, 2004, Trifunovic et al, 2005). Results indicated that despite the presence of severe respiratory chain dysfunction, the amount of ROS produced in these mice was normal, no increased sensitivity to oxidative stress-induced cell death was observed and no difference in oxidative damage to protein was seen. This data thus suggests that if ROS does increase with age, it may not be due to respiratory chain dysfunction.

An alternative source of ROS may come from age-dependent up-regulation of the inflammatory response. Inflammation has been implicated in many age-related diseases such as rheumatoid arthritis, osteoarthritis and cardiovascular disease (Licastro et al, 2005). The presence of inflammation is associated with increased ROS production, promoting the destruction of normal tissue (Winrow et al, 1993). This production of ROS during an inflammatory response may also initiate and/or amplify inflammation via the up-regulation of several genes involved in the pro-inflammatory response (Conner and Grisham, 1996). In some instances however, inflammation associated with ageing may result from direct damage from ROS (Chung et al, 2001). It is therefore difficult to determine to determine what come first, the inflammation leading ROS production or ROS production leading to damage leading to an inflammatory response and further ROS production.

One source of inflammation, as discussed later, may come from the presence of senescent cells within tissues which adopt a proinflammatory phenotype. It is hypothesised that the accumulation of senescent cells within tissues contributes to ageing (Hayflick, 1965). As the number of senescent cells increases, so does the intensity of the inflammatory response. In part, this proinflammatory phenotype is thought to damage tissues by the production of ROS.

Another alternative for increase in ROS with age may be due to a reduction in antioxidant defences and/or a decrease in the failure to repair oxidative damage.

Long-lived Proteins

The accumulation of damage on many proteins is most likely to have little or no effect on tissues if the proteins are constantly being turned-over. Damaged proteins would be removed and replaced, eliminating any detrimental affect such damage may have caused if it persisted. However, a number of studies have focused on proteasome damage as an ageing mechanism (Friguet, 2002, Farout and Friguet, 2006). The proteasome is the main proteolytic system responsible for protein degradation and is itself a protein. Therefore, accumulating damage to the proteasome may result in the non-removal and accumulation of damaged proteins which may subsequently have a detrimental impact on tissues. However, if the proteasome itself has a high turnover rate, then protein damage should have little if no affect overall. Data for proteasome turnover rates is lacking but one study found that many components which make up the proteasome display high turnover rates (Hayter et al, 2005). Since there is little evidence to suggest accumulated protein damage in these proteins has any impact on age-related tissue dysfunction, more focus will be given to proteins with low rates of turnover.

Long-lived proteins are commonly extracellular and normally involved as structural components of tissues, and these includes collagen, the most abundant protein in the body found in skin, bones, tendons and teeth. Crystallin is also a long-lived protein found in the lens of the eye. Since these proteins have a very low turnover rate they are more likely to accumulate, as yet, irreversible damage and thus impair tissue function. One study investigated turnover rates of collagen and calculated the half-life of cartilage collagen to be over 100 years and that of skin to be 15 years (Verijl et al, 2000).

Damage may result in chemical modifications resulting in structural changes to the proteins and consequently altering its interactions with other proteins. One of these chemical modifications which have been researched extensively in regard to ageing and disease are the formation of advanced glycation end-products (AGEs). AGEs are formed when reducing sugars such as glucose or fructose react spontaneously with lysine or arginine residues in proteins (Wautier and Schmidt, 2004). The formation of AGEs can result in the cross-linking of proteins such as collagen and lens crystalline. Since these proteins are functionally different, the biological impact of protein cross-linking is going to be different. Cross-linking of collagen may result in arterial and joint stiffening whereas the formation of cataracts may be observed in lens crystalline.

The accumulation of AGEs in cartilage affects not only biochemical but also biomechanical and cellular properties of the tissue (Verzijl et al, 2003). At the biomechanical level, the accumulation of AGEs results in increased stiffness of the tissue and increased brittleness of the cartilage collagen network, increasing the risk of mechanical damage. Changes at the cellular level in response to AGE accumulation include decreases in proteoglycan and collagen synthesis by chondrocytes and decreased susceptibility of the cartilage matrix components towards proteinase-mediated degradation. Both these cellular alterations suggest that chondrocytes in a glycated environment have a reduced capacity to remodel their matrix and as a result reduce the capacity of chondrocytes to repair damage.

Since glucose is needed for the production of AGEs, their accumulation and consequently increasing stiffness is most likely to be proportional to blood glucose levels and the length of time these persist. Therefore, metabolic disorders such as diabetes mellitus in which blood glucose are often high would be affected more severely by AGEs compared with normal ageing. Arterial stiffness has been shown to be greatly accelerated in patients suffering from type 1 and type 2 diabetes (Schram et al, 2002, 2003). The exact mechanisms resulting in arterial stiffness are currently unknown. However, AGE formation leading to cross-linking of collagen and elastin and subsequent loss of elasticity is thought to be a key contributor. With this in mind, one study used ALT-711, a breaker of AGE crosslinks and determined whether arterial compliance was improved (Kass et al, 2001). Results showed that subjects treated with ALT-711 displayed an improvement in arterial compliance in aged humans with vascular stiffening. Arterial compliance rose 15% in ALT-711 treated subjects compared with no change with placebo. Similar experiments on aged dogs found that after 1 month of treatment with ALT-711, a significant reduction (~40%) in ventricular stiffness was observed and accompanied by improvement in cardiac function (Asif et al, 2000). These results suggest that AGEs are at least partly a contributor to arterial stiffness.

Another secondary effect of diabetes, which is possibly due to AGE formation is that seen with the development of cataracts (Ulrich and Cerami, 2001). Human lens crystallins are important long-lived proteins involved in retaining optical clarity required for normal vision. It is possible that glucose and other substances modify lens crystallins, causing conformational changes which subsequently result in the scattering of light and producing a cataract. One study investigated the occurrence of AGEs in human lenses and found a strong relationship between lens AGE content and the state of the cataract (Franke et al 2003). Another study compared AGEs in human diabetic and non-diabetic cataractous lenses and also found an overall increase of AGEs in diabetic lenses compared with non-diabetic lens samples (Pokupec, et al, 2003). Both these studies provide correlative support for the notion that AGEs play a causal role in cataracts.

In any biological system the structure-function relationship is essential for normal activity. These two examples demonstrate how a change in structure can have a detrimental impact on normal biological function. It also demonstrates how one ageing mechanism can have multiple consequences depending on the tissue in question.

More recently, findings have suggested that AGE formation may not only affect the structure-function of long-lived proteins but may also have an impact on cellular activities. This idea comes from studies that have demonstrated the interaction of AGEs with specific cell surface receptors, of which the best characterised receptor is RAGE (receptor for AGE). RAGE is a cell surface receptor present on different cell types including endothelial cells, smooth muscle cells, lymphocytes and macrophages (Wautier and Guillausseau, 1998). The binding of AGEs to specific receptors is thought to lead to cellular activation, increased expression of extracellular matrix proteins and the release of pro-inflammatory cytokines and growth factors (Simm et al, 2004). Therefore, the interaction of accumulating AGEs with receptors may cause undesirable changes in cell function, which may in turn affect the functioning abilities of tissues.

Mechanisms of Ageing

When discussing mechanisms of ageing it is important to distinguish between the different components which make up living tissue, as the predominant ageing mechanism for each is going to be different. It is unlikely that the ageing process is governed by one universal mechanism. Human tissue is composed of a mixture of long-lived structural proteins (such as crystallin and collagen), post-mitotic cellular elements (such as mature neurones and muscle fibre cells) and mitotic cells (such as T-cells, endothelial cells and fibroblasts). The gradual biological alterations which drive the ageing process cause changes to occur in all the components of this complex mixture. However, the mechanisms by which each component degenerates are often distinct and since all these components interact with each other, a change in one will have a direct impact on another.
The main focus of ageing research is to prevent/combat age-related disease and disability, allowing everyone to live healthier lives for longer.