Why do senescent cells accumulate in tissues?

If the accumulation of senescent cells are so detrimental to the tissues in which they reside, why haven’t we evolved mechanisms to remove them? The answer is that we probably have, but the mechanism which removes them from the tissues becomes impaired as with age.

To understand how this removal system may work, we need to look at the phenotype of senescent cells. Although a large number of the changes which occur during cellular senescence may be cell specific, there appears to be features which are common to the majority of senescent cell types. These include the secretion of growth factors, matrix degrading proteins (MMPs) and the production of cytokines. Since these factors are a common feature, it is likely that they have a common function and are not just a random consequence of the changes which occur during senescence.

One possibility is that senescent cells are removed by the immune system. Senescent cells secrete cytokines to attract immune cells to their location (for their removal), secrete matrix degrading proteins to allow the immune cells easy access and secrete growth factors to stimulate the proliferation of surrounding cells for its replacement once the cell is removed. However, since the immune system itself is governed by ageing mechanisms, its ability to remove senescent cells gradually decreases, therefore the accumulation of senescent cells gradually increases.

The majority of the work on the immune clearance of unwanted cells has been carried out in cancer research. The prevalence of cancer as we all know increases with age, and this may be due to an ageing immune system, consequently resulting in an impaired ability to remove cancer cells as they appear. Over the past several years it has become clear that the immune system plays a crucial role in preventing cancer. As a consequence, there has been a great deal of interest in using our bodies own immune system to recognise and destroy cancer cells (FDA, cancer research uk), a process which could potentially be used to target and destroy senescent cells in ageing tissues.

Publication

DGA Burton (2008) Cellular senescence, ageing and disease. AGE


Would it be a disaster to find a cure for ageing and death by natural causes?

Richard Faragher presented a light-hearted and often amusing talk on the question “Would it be a disaster to find a cure for ageing and death by natural causes?” This talk was part of the Big Questions series held at the University of Derby (UK) which allows scientists to discuss various science-based issues with people of faith (and of no faith). This talk was recorded for a podcast for all to enjoy.

Richard begins his talk with a brief background, explaining what ageing is, the theories behind why we age and a discussion of the current theories of how we age. He then goes on to talk about whether immortality is actually possible, finishing with religious perspectives on ageing research.

During his talk, Richard discusses data which demonstrates that life expectancy is increasing, but healthy life expectancy is not increasing as fast. With this in mine, he talks about how research into ageing will help increase the rate of healthy life expectancy. This he does by mentioning research using animal models which alter the rate of ageing and the resulting decreases in both the rate at which pathology appear and their severity once present. He goes on to talk about promising research by a team headed by Janet Lord at the University of Birmingham (UK), and how such research has the potential to save tens of thousands of lives a year, thus providing an excellent example of how research into the biology of ageing can help people.

Not often discussed, but a much needed mention in regard to ageing research, especially to those new to the area, are those people who are detrimental to the reputation of ageing research. Richard puts these into three classes of people:

(1) Scientists who say off-the-cuff statements which consequently get blown out of proportion by the media (i.e. “Scientists find cure for ageing”). Ageing is not cured.

(2) Those who say ageing is cured, for the purpose of making money. i.e. from immortality devices and anti-ageing pills.

(3) Visionaries (or “persuasive prophets”) which tell of things to come (i.e. Living to 1000), gaining valuable media attention which should be spent for those doing proper ageing research.

The final discussion on religious perspectives of ageing research is something I’ve never encountered before and definitely worth a listen.

So what you waiting for?...get listening (click here)


http://scipodem.wordpress.com/

Blog entries over the coming weeks is going to be sparse as I finally need to concentrate my efforts on my Phd Thesis…..but stay tuned.

Why is cyclin D1 upregulated during cellular senescence?

The abstract below is from a recent publication of mine which shows my recent findings on the search for biomarkers of cellular senescence, which formed part of my PhD. It presents data which suggests that cyclin D1 overexpression can be used to detect senescent vascular smooth muscle cells and fibroblasts. However, there is little discussion on why cyclin D1, a known protein involved in cell cycle progression, is up-regulated at senescence (a state of growth arrest). The following is a literature review which may provide insight into why cyclin D1 is up-regulated in some senescent cell types.

Cyclin D1 Overexpression Permits the Reproducible Detection of Senescent Human Vascular Smooth Muscle Cells

The senescence of mitotic cells is hypothesized to play a causal role in organismal aging. Cultures of normal human cells become senescent in vitro as a r
esult of a continuous decline in the mitotic fraction from cell turnover. However, one potential barrier to the evaluation of the frequency and distribution of senescent cells in tissues is the absence of a panel of robust markers for the senescent state. In parallel with an analysis of the growth kinetics of human vascular smooth muscle cells, we have undertaken transcriptomic comparisons of early- and late-passage cultures of human vascular smooth muscle cells to identify potential markers that can distinguish between senescent and growth-competent cells. A wide range of genes are upregulated at senescence in human vascular smooth muscle cells. In particular, we have identified a 12-fold upregulation of expression in the cyclin D1 message, which is reflected in a concomitant upregulation at the protein level. Quantitative cytochemical analysis of senescent and growing vascular smooth muscle cells indicates that cyclin D1 reactivity is a considerably better marker of replicative senescence than senescence-associated β-galactosidase activity. We have applied this new marker (in combination with Ki67, COMET, and TUNEL staining) to the study of human vascular smooth muscle cells treated with resveratrol, a putative anti-aging molecule known to have significant effects on cell growth.

An understanding of why cyclin D1 is up-regulated at senescence may provide further insight into the molecular pathways governing cellular senescence (and cancer).

Interestingly, two variants of cyclin D1 exist (Solomon et al, 2003), these variants, designated cyclin D1a and cyclin D1b have been shown to differ in their behavior. Cyclin D1b does not possess the Thr286 phosphorylation site required for nuclear export and regulated degradation (Knudsen, 2006). As a result, the cyclin D1b protein appears to be constitutively localised in the nucleus, whereas cyclin D1a is exported to the cytoplasm during S-phase. Despite enhanced nuclear localisation, it was found that cyclin D1b is a poor regulator of RB phosphorylation/inactivation.

Probably the most interesting findings on cyclin D1 up-regulation come from Berardi et al (2003). This group identified a novel transcriptional regulatory element in the 5’-untranslational region of the cyclin D1 gene that differentially suppresses cyclin D1 expression in young versus senescent fibroblasts. Abundant protein complexes were found to be forming with young cell nuclear extracts compared with senescent cells nuclear extracts and binding was maintained in quiescent cells, showing that loss of activity was specific to senescent cells and not an effect of cell cycle arrest. These findings thus suggest that loss of transcriptional repressor activity may contribute to the up-regulation of cyclin D1 during cellular senescence.

Alt et al (2002) suggests that the accumulation of cyclin D1 at senescence may be due to elevated levels of p21. Evidence suggests that p21 promotes nuclear accumulation of cyclin D1 complexes via inhibition of cyclin D1 nuclear export. However, another study has demonstrated that oncogenic Ras promotes the accumulation of p21 by elevating the levels of cyclin D1 (Coleman et al, 2003). Colman and co-workers also found that this increase in cyclin D1 was sufficient to inhibit proteasome-mediated p21 degradation. Knock-down of cyclin D1 by RNA interference confirmed that RAS-induced p21 stabilisation was dependent upon cyclin D1 expression. They also showed that p21 directly binds to the C8α subunit of the 20S proteasome complex and that by competing for binding, cyclin D1 inhibits p21 degradation by purified 20S complexes in vitro. They therefore proposed that Ras stabilises p21 by promoting the formation of p21-cyclin D1 complexes that prevent p21 association with, and subsequently degradation by, the 20S proteasome. In some circumstances, the activation of Ras leads to cell cycle arrest similar to that observed with replicative senescence (Mason et al, 2004). It is therefore possible that at the onset of cellular senescence p21 is elevated, this in turn promotes nuclear accumulation of cyclin D1 which stabilises p21 and allowing it to accumulate further.

It was mentioned previously that the cyclin D1b variant lacks the Thr286 phosphorylation site required for nuclear export and degradation and is a poor catalyst for pRb phosphorylation. It is possible that during the replicative lifespan of a cell, this cyclin D1b variant could gradually accumulate within the cell nucleus binding and stabilising p21 until it reaches a threshold where all cyclin D1-Cdk complexes have bound to p21, triggering growth arrest. However, it has been reported that this cyclin D1 variant does not accumulate in cells and exhibits stability comparable with cyclin D1a (Soloman et al, 2003).

Senescent cells that express high levels of cyclin D1 are unable to phosphorylate pRb in response to mitogenic stimuli (Atadja et al, 1995). This study showed that the lack of pRb phosphorylation at senescence occurred when virtually all cyclin D1-Cdk complexes became associated with p21 (Dulic et al 1993). Therefore, it seems that low levels of cyclin D1 play a positive role in cell cycle progression by phosphorylating and thus neutralising the inhibitory activity of pRb. However, when cyclin D1 is elevated, it has a negative effect on the cell cycle by stabilising p21 and inhibiting cyclin D1-Cdk complexes from phosphorylating pRb, resulting in cell cycle arrest.

Increased p21 expression appears to only initiate telomere dependent senescence, but later, the senescent state is maintained by p16, at which point p21 is down-regulated (Stein et al, 1999). If p21 is down-regulated, this may also result in the down-regulation of cyclin D1.

If p21 expression is required to stabilise and consequently up-regulate cyclin D1, is cyclin D1 up-regulated in cells which are not dependent upon telomere shortening in order to senesce? Telomere independent senescence appears to trigger the up-regulation of p16 alone. This would suggest that cyclin D1 cannot be detected in these cell types. Interestingly, Opitz et al (2001) found that cyclin D1 overexpression alone was enough to extend the replicative lifespan of normal oral keratinocytes, a cell type known to senesce by telomere-independent mechanisms. Therefore, cyclin D1 overexpression in these cells have the opposite impact on cell state.



Typical dual staining of MRC-5 fibroblasts with cyclin D1 (FITC, green) and Ki67 (TRITC, red). DAPI (counterstain) is blue.

Ageing: Molecules to Man

British Society for Research on Ageing (BSRA) is to hold its annual scientific meeting in Brighton, July 17-18, 2008. The theme is "Ageing: Molecules to Man."

"Our meeting takes a multidisciplinary approach to one of the key problems in ageing today: the concept of frailty, says Richard Faragher, BSRA Secretary. The speakers programme combines established leaders in the fields of calorie restriction, muscle biology and cellular senescence with some of the best and brightest newcomers to ageing research.

Confirmed speakers include:

Paul Thornalley, University of Warwick (the chemistry of ageing)
Chris Moulin, University of Leeds (the ageing mind)
• Calvin Harley, Geron Corporation (replicative senescence)
David Gems, University College London (ageing in invertebrates)
Arlan Richardson, University of Texas San Antonio (rodent models of ageing)
• Steve Allen, Bournemouth (respiratory frailty and ageing)

The meeting will be held at the Hilton Brighton Metropole in a prime seafront location, in the heart of Brighton.

The meeting hopes to capture the international quality and reach of British ageing research. It is also designed to promote networking and collaboration for attendees from around the world. Certificates of attendance will be available for CPD purposes.

www.seniorsworldchronicle.com

www.bsra.org.uk

The need for an effective biomarker of senescent cells

An effective biomarker of cellular senescence is required so senescent cells can be visualised both in vitro and in vivo, allowing their frequency and distribution to be monitored in ageing and diseased tissues.

At present, the most commonly used method to detect senescent cells is a modified beta-galactosidase assay (Dimri et al, 1995). Detectable β-galactosidase at pH 6 was found to increase during replicative senescence of fibroblast cultures in vitro and in vivo and was absent in immortal cell cultures. This was termed senescent-associated β-galactosidase or SA-β-Gal. However, since this first report, there have been numerous studies that have demonstrated SA-β-Gal staining in non-senescent cells.

For example, it has been reported that SA-β-Gal activity is detectable in quiescent cultures of Swiss 3T3 as well as some types of human cancer cells that were chemically stimulated to differentiate (Yegorov et al, 1998). After 21 days in culture, Swiss 3T3 cells in low serum displayed 40-50% SA-β-Gal positive cells and cells treated to differentiate after 13 days displayed as high as 75% staining. Another study looked at the expression of SA-β-Gal in human ovarian surface epithelial cells (HOSE 6-3) undergoing immortalisation by the human papilloma viral oncogene E6 and E7 (Litaker et al, 1998). They found that HOSE 6-3 cells expressing SA-β-Gal was highest (39%) when cells were at crisis. After this stage when cells achieved immortalisation status SA-β-Gal activity sharply decreased (1.3%).

Severino et al (2000) specifically focused on determining the robustness of SA-β-Gal activity as a marker of replicative senescence . This study characterised changes in SA-β-Gal staining in a variety of different conditions. SA-β-Gal activity was found to be elevated in confluent non-transformed fibroblast cultures, in immortal fibroblast cultures that had reached a high cell density and in low-density young, normal cultures oxidatively challenged by treatment with H2O2. They concluded that although SA-β-Gal staining is increased under a variety of different conditions, the interpretation of increased staining remains unclear.

SA-β-Gal staining has also been shown to be a marker for differentiation of human prostate epithelial cells (HPEC) (Untergasser et al, 2003). HPEC cells stimulated with transforming growth factor beta (TGF-β), resulted in an increase in SA-β-Gal activity but showed no terminal growth arrest nor induction of important senescent-associated genes such as p16. It was therefore suggested that TGF-β could contribute to the increased number of SA-β-Gal positive epithelial cells observed in benign prostatic hyperplasia (BPH).

A recent report demonstrated that fibroblasts from patients with autosomal recessive G(M1)-gangliosidosis, which have defective lysosomal beta-galactosidase did not express SA-β-Gal at late passage even though they underwent replicative senescence (Lee et al, 2006). It was also demonstrated that cells depleted of GLB1 (the gene encoding lysosomal beta-D-galactosidase) mRNA underwent senescence but failed to express SA-β-Gal. SA-β-Gal activity is therefore dependent upon lysosomal mass rather than growth state. If this is indeed the case, SA-β-GAL staining would most likely underestimate the percentage of senescent cells in a sample.

DISEASE FOCUS: Atherosclerosis and vascular calcification

Cellular Senescence and vascular calcification
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Data suggesting that cellular senescence plays a role in vascular calcification was first presented by myself (as far as I am aware, please let me know otherwise) at the Integrative Physiology Post-Graduate Conference, Aberdeen (2007). The abstract was as follows:

A transcriptomic analysis of vascular smooth muscle cells

The senescence of mitotic cells is thought to play a role in ageing and age-related disease. To investigate this further, RNA was extracted from growing and senescent cultures of vascular smooth muscle cells (VSMCs) and subjected to microarray analysis. A literature search of genes involved in atherosclerosis and vascular calcification (an age-related vascular disease) was undertaken and the expression of those genes investigated using the microarray data of senescent VSMCs. Results show that genes known to be involved in atherosclerosis and vascular calcification are significantly up or down regulated in senescent VSMC. ELISA and Western blot analysis was used to validate the microarray data. These results suggest that senescent VSMCs play a role in the development and/or the progression of atherosclerosis and for the first time suggest a role in vascular calcification.


Data from this study (soon to be published) shows that those proteins which are either up or down-regulated at sites of calcification are also transcriptionally up or down-regulated in cultures of senescent vascular smooth muscle cells (VSMCs). The main two culprits involved in calcification appear to be matrix gla protein (MGP) and bone morphogenic proteins (BMP). MGP is normally expressed in endothelial cells and has been identified as a calcification inhibitor of the arterial wall and is thought to neutralise the known effects of BMPs (Zebboudj et al, 2002). In contrast, BMPs are important anabolic factors in bone formation and determinant of bone mineral content (Garrett et al, 2007).

In cultures of senescent VSMC, MGP expression is down-regulated 24-fold (the largest down-regulation of any gene on the chip (affymetrix)), whereas BMP2 is up-regulated more than 4-fold. Since control of BMP activity is important for normal bone formation, the up-regulation of BMPs in senescent VSMC (and the down-regulation of its inhibitor, MGP), suggests senescent VSMC play an important role in the pathophysiology of vascular calcification. BMP2 may be responsible for inducing osteoblastic differentiation of vascular smooth muscle cells, a process thought critical in the initiation of vascular calcification (Hruska et al 2005).
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To further demonstrate the importance of MGP in preventing vascular calcification, MGP knock-out studies were carried out on mice (Luo et al, 1997). Mice lacking MGP died within a few months as a consequence of arterial calcification which lead to blood-vessel rupture. However, in calcified arteries, MGP expression has been found to be up-regulated (Mazzini and Schule, 2006), but this is probably an attempt (by non-senescent cells) to reduce the levels of calcification resulting from uncontrolled expression of BMP2.
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In atherosclerosis, calcification can occur in advanced lesions. Stimulated proliferation of VSMC in developing plaques reduces the replicative capacity of those cells and increase the appearance of senescence cells. These senescent VSMC may up-regulate BMPs and down-regulate MGP, thus resulting in calcification.

Although, much more work is required to validate the microarray data and investigate these findings in living tissues, this preliminary work suggests for the first time that senescent VSMC may play a role in the development/progression of vascular calcification.

DISEASE FOCUS: Atherosclerosis and vascular calcification

Vascular calcification

Vascular calcification is a prominent feature of advanced atherosclerotic lesions. Vascular calcification refers to the deposition of calcium phosphate mineral in the intima or media of arterial walls, leading to reduced elasticity and compliance. The mechanism underlying vascular calcification is currently unknown. However, a number of studies have suggested that the process of vascular calcification is similar to the mineralisation process observed in bone (Abedin et al, 2004). This is based on the observation that bone-associated proteins such as osteocalcin, osteonectin, bone morphogenic proteins (BMP) and matrix Gla proteins (MGP) have been detected in vascular calcifications (Trion et al, 2004). VSMC appear to be an important factor in vascular calcification, since VSMC within calcified plaques have been shown to express osteoblast and chondrocyte-like gene expression profiles (Tyson et al, 2003). MGP, osteonectin, osteprotergerin and aggrecan were constitutively expressed by VSMC in normal arteries but were found to be down-regulated in calcified arteries. Since MPG has been shown to inhibit calcification, its down-regulation observed in these plaques may be the key factor in initiating vascular calcification. Little is known about the mechanisms governing vascular calcification.

DISEASE FOCUS: Atherosclerosis and vascular calcification

Inflammation and atherosclerosis

Atherosclerosis was once considered to be predominantly a lipid storage disease but mounting evidence suggests that inflammation is critical at every stage, from initiation to progression and eventually plaque rupture (Paoletti et al 2004).

Inflamed endothelial cells in the lining of arteries release pro-inflammatory cytokines which provide a chemotactic stimulus to adhere leukocytes and monocytes, directing their migration into the intima (Boisvert, 2004). These inflammatory cells release pro-inflammatory mediators responsible for differentiating monocytes to lipid-laden macrophages, foam cells (Frostegard et al 1999). These foam cells also secrete proinflammatory cytokines that amplify the local inflammatory response in the lesion (Libby, 2002). The secretion of cytokines and growth factors stimulate the migration and proliferation of SMC. These cytokines also stimulate the secretion of matrix degrading proteins from SMC which permits the penetration of SMC through the elastic laminae and extracellular matrix (ECM) of the growing plaque. Inflammatory mediators can inhibit ECM protein synthesis and increase expression of matrix degrading proteins by foam cells within the intimal lesion (Libby, 2002). Since the strength of the plaques fibrous cap is due to the extracellular matrix, its degradation would result in loss of strength and increased chance of rupture.
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Cellular senescence and atherosclerosis
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It has been suggested that injury to endothelial cells results in endothelial dysfunction which may lead to the development of atherosclerotic plaques (Kitamoto and Egashira, 2004). How this initial damage to endothelial cells occurs is currently speculative, but there is increasing evidence to postulate that this initial endothelial dysfunction may be the result of cellular senescence.

Early histological studies of advanced human atherosclerotic lesions suggested the presence of senescent endothelial cells (Burrig et al, 1991). Endothelial cells exhibiting the morphological features of senescence were frequently found on the plaque surface. The presence of senescent cells within plaques was also found in studies of vascular cells in culture, derived from human atherosclerotic plaques (Bennett et al 1998). VSMC derived from atherosclerotic plaques were shown to have lower rates of proliferation and underwent senescence earlier than cells derived from normal vessels. With the emergence of a biomarker (SA-β-Gal) which could detect senescent cells in vivo, a more direct approach for investigating cellular senescence in diseased tissue was undertaken (Fenton et al, 2001). This study sought to detect the presence of senescent cells in injured rabbit carotid arteries. Results indicated the accumulation of senescent cells in the neointima and media of all injured vessels, in contrast to the near absence of such cells in control vessels. Similar investigations have also been carried out on human atherosclerotic plaques (Vasile et al 2001, Minamino et al 2002). Both these studies demonstrated the presence of senescent vascular endothelial cells in vivo at sites of atherosclerotic plaque formation as detected by SA- β-Gal.

More recently due to advances in molecular biology, there have been numerous investigations involving the biology of telomeres in atherosclerosis. One such study examined telomere length in cells from atherosclerotic plaques and normal vessels and demonstrated that VSMC from plaques had markedly shorter telomeres compared with normal VSMC (Matthews et al 2006). This shortening was found to be closely associated with increasing severity of atherosclerosis. As with previously mentioned studies, these VSMC demonstrate morphological features of senescence when cultured in vitro. A similar study investigated telomere lengths of endothelial cells (EC) from coronary artery disease (CAD) and also found that telomeres were significantly shorter in CAD compared with normal arteries (Ogami et al, 2004). Since both VSMC and EC of atherosclerotic plaques have been shown to have senescent cells present and cellular senescence is generally attributed to the attrition of telomeres, the presence of cells with shorter telomeres in these tissues is therefore not surprising.

The above studies provide evidence for the presence of senescent cells in atherosclerotic plaques, but provide little explanation for their occurrence. This is less true for senescent SMC, since their presence can be explained by stimulated proliferation and migration observed in atherosclerosis. SMC have a finite replicative capacity, most likely as a result of telomere shortening, therefore, constant rounds of cell division would eventually result in the cell becoming senescent. The presence of senescent SMC would therefore only be observed in late stage plaque development, since this is when SMC are stimulated to migrate and proliferate.

Since the initiation of plaque development begins at EC, an explanation for why there may be senescent cells present is harder to explain. One possibility is that senescent EC cells in atherosclerotic plaques is most likely due to proliferative exhaustion as a result of replacing lost and damaged cells. As previously discussed, plaque formation is commonly seen within arteries at areas of high shear stress. It is therefore possible that such high shear stress could lead to the loss of ECs in these areas, which subsequently need to be replaced. This would result in an increase in cell turnover at those sites and consequently the occurrence of senescent EC. Since senescent cells in general can be classed as dysfunctional, it may be the presence of senescent EC within the endothelium which is the initiating factor in plaque formation. Further evidence for this may be provided if the expression profile of senescent vascular ECs were compared with vascular ECs of lesion-prone sites within arteries.

Since the recruitment and accumulation of leukocytes and monocytes is an important step in the development of atherosclerosis, the expression of proteins such as intracellular adhesion molecule-1 (ICAM1) and vascular cell adhesion molecular-1 (VCAM-1), important mediators of leukocyte and monocyte adherence have been investigated. One study looked at the expression of VCAM-1 and ICAM-1 at lesion-prone sites on the endothelium in ApoE-deficient mice (which are more prone to lesion development) (Nakashima et al, 1998). Staining for VCAM-1 showed localised staining at lesion-prone sites in ApoE-/- mice and only weak staining limited to sites of altered blood flow in control mice. ICAM-1 was the most prominent adhesion molecule in lesion prone sites and was up-regulated in ApoE -/- mice and control mice. If ICAM-1 is being up-regulated as a result of senescent cell formation, it is not surprising that it is found up-regulated in both ApoE -/- and control mice, since the same high shear stress is most likely occurring in both mice. Another study specifically investigated whether endothelial dysfunction was the result of endothelial cell senescence by inducing senescence in human aortic endothelial cells (HAECs) and examining the expression of ICAM-1 and endothelial nitric oxide synthase activity (eNOS) (Minamino et al, 2002). Results showed that ICAM-1 expression was increased and eNOS activity decreased in senescent HAECs. There are numerous studies that have shown eNOS activity to be decreased during endothelial dysfunction and this decrease is thought to play a critical role in the development and progression of atherosclerosis (Yang et al, 2006). Up-regulation of ICAM-1 and a decrease in eNOS activity in both senescent endothelial cells and at lesion-prone sites, strongly suggests that senescent cells are a significant contributing factor in initiation of atherosclerotic plaque formation.

DISEASE FOCUS: Atherosclerosis and vascular calcification

Overview of atherosclerosis

Cardiovascular disease accounts for approximately 56% of the total mortality in the over 65 age group and represents the single largest age-related cause of death (Brock et al, 1990, Mills et al). Atherosclerosis constitutes the single most important contributor to this increasing problem of cardiovascular disease. Atherogenesis is a complicated process which includes endothelial cell (EC) dysfunction, smooth muscle cell (SMC) proliferation and migration, recruitment of inflammatory cells, lipid and matrix accumulation and thrombus formation (Tuomisto et al 2005).

To better understand the pathological processes that occur with atherosclerosis, an understanding of the structure of arteries is required. Human arteries are composed of three layers, the intima, the media and the adventitia. The intima is the innermost layer of the artery, composed of EC’s, SMC’s, macrophages and extracellular matrix (ECM) components. An internal elastic lamina separates the intima from the media, which is made up mainly of SMC. The adventitia is separated from the media by external elastic lamina and is mainly composed of fibroblasts and connective tissue.

The initiation and progression of atherosclerotic plaques generally takes place over many years during which the affected individual remains symptom free. Therefore, when a patient becomes symptomatic, the disease is already well established. These plaques occur at specific sites within arteries and these sites are dictated by fluid shear stress, the frictional force generated by blood flow over the vascular endothelium (Hwang et al, 2003). Regions of branched and curved arteries experience the greatest disturbed blood flow and it is at these sites that high incidences of plaque formation is found (VanderLaan et al, 2004). Relatively straight arteries however, experience the least shear stress and are usually protected from plaque development. Explanations for why high fluid stress sites are more “lesion-prone” is currently speculative.

The initial factors which result in the initiation of plaque formation are currently unknown. A common hypothesis is that plaque formation occurs as a result of EC damage leading to cellular dysfunction (Shimokawa, 1999, Davignon and Ganz, 2004). The source of the initial damage to EC’s is also currently unknown, but hypertension, viruses, toxins, smoking have all been suggested. Cellular dysfunction results in subsequent recruitment and accumulation of leukocytes and monocytes which would otherwise have resisted any adhesive interactions (Bobryshev et al, 2005). These adhered monocytes then differentiate into macrophages, engulf lipids, become foam cells and form fatty streaks. As the progression of the plaque continues, SMC’s migrate from the intima and synthesis extracellular matrix proteins in the intima (Boyle et al, 1997). Progressive macrophage accumulation, SMC migration and proliferation and extracellular matrix protein synthesis result in the formation of an advanced lesion.


A schematic representation of the structure of an artery, showing the intima, media and adventitia (commons.wikimedia.org)

Phenotypic changes associated with cellular senescence

When a cell becomes senescent, changes at the genetic level occur which subsequently has an effect on both cell behaviour and morphology. Microarray analysis of senescent dermal fibroblasts, retinal pigment epithelial cells and vascular endothelial cells demonstrate overlap in gene expression changes but overall display cell-type specific changes (Shelton et al, 1999). Similar studies were carried out looking at human dermal fibroblasts and oral keratinocytes (Yoon et al, 2004, and Kang et al, 2003). These studies found transcriptional changes in genes associated with inflammation, regulation of cell cycle, cytoskeletal genes and extracellular matrix (ECM) genes. More recently, microarray analysis of primary human lung fibroblasts (IMR-90) and primary skin fibroblasts (Detroit 551) reported that out of the of the 4183 genes analysed, 165 were down-regulated and 191 up-regulated in senescent IMR-90 cells and 154 down-regulated and 76 up-regulated in senescent Detroit 551 cells compared with their growing counterparts (Chen et al, 2004). This degree of alteration to the transcriptome is akin to that seen when cells are induced to differentiate (Truckenmiller et al, 2001; Gerhold et al, 2002).

Impairment in cell mobility, secretion of matrix degrading proteins, secretion of growth factors and pro-inflammatory cytokines are considered as significant changes associated with cellular senescence. All these factors have the potential to cause detrimental damage to tissues.

A number of papers have reported that the ability of senescent cells to migrate is severely reduced (Schneider and Mitsui, 1976; Sandeman et al, 2000; Reed et al, 2001). This decline in the ability to migrate may be related to changes which occur to the cytoskeleton during cellular senescence (Nishio and Inoue, 2005). Actin is an important component of the cytoskeleton required for cellular migration. However, in senescent fibroblasts for example it has been shown that vimentin is produced in place of actin which is down-regulated. This migration deficit has important implications during wound healing since cells are stimulated to migrate into the wound, proliferate and construct the new extra-cellular matrix (ECM). Also, since senescent cells tend to secrete proteins which degrade the matrix, wound repair would be impaired.

Matrix metalloproteases (MMPs) are also commonly up-regulated in senescent cells (Sandeman et al, 2001, Campisi, 2005). In normal tissue processes, MMPs are required for fertilization, cellular adhesion, development, neurogenesis, and metastasis (Page-McCaw et al, 2007). However, MMP secretion by senescent cells has also been suggested to play a role in the progression of disease such as in the pathogenesis of coronary heart disease (CAD) (Nanni et al, 2007). MMPs have also been implicated in the progression of osteoporosis, since MMPs play important roles in bone resorption (Logar et al, 2007). One study has also shown that the secretion of MMPs by senescent chondrocytes may contribute to the development or progression of osteoarthritis (Price et al, 2002).

Abnormal secretion of some growth factors has been shown to be another general characteristic of senescent cells. Work on human fibroblasts found that vascular endothelial growth factor (VEGF) secretion is elevated in senescent cell cultures (Coppe et al, 2006). Since growth factors are capable of stimulating cellular proliferation it has been suggested that while initially cellular senescence may be a mechanism to suppress tumourigenesis early in life it may promote cancer in aged organisms (Campisi, 1997). Human senescent fibroblasts for example have been shown to stimulate premalignant and malignant, but not normal epithelial cells to proliferate in culture and form tumours in mice (Krtolica et al, 2001, Krtolica and Campisi 2002). Another study sought to characterise the molecular alterations that occur during prostate fibroblast senescence to identify factors which may be capable of promoting the proliferation and potentially the neoplastic progression of prostate epithelium (Bavik et al, 2006). Fibroblast growth factor 7 (FGF7), hepatocyte growth factor and amphiregulin (AREG) were found to be elevated in the extracellular environment of senescent prostate fibroblasts. Direct co-culture and conditioned medium from senescent prostate fibroblasts stimulated epithelial cell proliferation 3-fold and 2-fold respectively. These results suggest that senescent cells may contribute to the progression of prostate neoplasia by altering the prostate microenvironment.

Probably the most potentially detrimental changes which can occur when cells become senescent is that of secreted cytokines since they not only effect local tissue but can have much wider impacts throughout the organism. Enhanced inflammation during ageing is thought to contribute to many of the diseases of ageing.

Vascular smooth muscle cells (VSMC) that have become senescent due to the activation of ras have been shown to drastically increase the expression of pro-inflammatory cytokines (Minamino et al, 2003). IL1α was shown to be up-regulated 11-fold, IL1β 50-fold, IL-6 12-fold and IL-8 77-fold. With such dramatic changes, it was suggested that this proinflammatory phenotype may contribute to the progression of atherosclerosis.

Senescent T-cells in vivo have been shown to produce high levels of two cytokines, IL6 and TNFα (Effros, 2004). Interestingly, the up-regulation of TNFα by T-cells in the bone marrow has been implicated as a causal mechanism in bone loss (Roggia et al, 2001).

Replicative senescence of human hepatic stellate cells (a major cell type involved in liver fibrosis) in culture also display a higher expression of inflammation genes (Schnabl et al, 2003). Interleukin-8 is among the cytokines up-regulated in senescent stellate cells (SC) which correlates with increase expression observed with disease activity in human alcoholic liver fibrosis (Sheron et al, 1993). Interleukin-6 is a known fibrogenic cytokine which was also shown to be up-regulated in SC senescent cultures. In normal conditions, chronic tissue damage results in the activation of SC characterised by proliferation, motility, contractility and synthesis of extracellular matrix (Gutiérrez-Ru, 2002). Since SC are stimulated to proliferate in response to tissue damage, the replicative capacity of these cells will be reduced and the accumulation of senescent cells accelerated. This activation of SC in response to tissue damage is regulated by cytokines and growth factors. Therefore, unregulated secretion of pro-inflammatory cytokines and growth factors from senescent SC within the liver may cause further damage. One study has shown for example that replicative senescence does have a significant impact in the long-term progression of fibrosis (Trak-Smayra et al, 2004).

This pro-inflammatory phenotype may partly be due to the up-regulation of intercellular adhesion molecule-1 (ICAM-1), a molecule known to be involved in inflammatory response and is over-expressed in senescent cells and aged tissues (Gorgoulis et al, 2005). One study has shown that p53 can directly activate the expression of ICAM-1 (Kletsas et al, 2004) and since p53 is activated and up-regulated during cellular senescence, it may activate ICAM-1, thereby contributing to the pro-inflammatory phenotype of senescent cells.
The main focus of ageing research is to prevent/combat age-related disease and disability, allowing everyone to live healthier lives for longer.