Ageing Research

Senescence surveillance of pre-malignant hepatocytes limits liver cancer development

2011-11-14T21:07:00.004Z

ABSTRACT

Tae-Won Kang, Tetyana Yevsa, Norman Woller, Lisa Hoenicke, Torsten Wuestefeld, Daniel Dauch, Anja Hohmeyer, Marcus Gereke, Ramona Rudalska, Anna Potapova, Marcus Iken, Mihael Vucur, Siegfried Weiss, Mathias Heikenwalder, Sadaf Khan, Jesus Gil, Dunja Bruder, Michael Manns, Peter Schirmacher, Frank Tacke, Michael Ott, Tom Luedde, Thomas Longerich, Stefan Kubicka & Lars Zender

Upon the aberrant activation of oncogenes, normal cells can enter the cellular senescence program, a state of stable cell-cycle arrest, which represents an important barrier against tumour development in vivo1. Senescent cells communicate with their environment by secreting various cytokines and growth factors, and it was reported that this ‘secretory phenotype’ can have pro- as well as anti-tumorigenic effects2, 3, 4, 5. Here we show that oncogene-induced senescence occurs in otherwise normal murine hepatocytes in vivo. Pre-malignant senescent hepatocytes secrete chemo- and cytokines and are subject to immune-mediated clearance (designated as ‘senescence surveillance’), which depends on an intact CD4+ T-cell-mediated adaptive immune response. Impaired immune surveillance of pre-malignant senescent hepatocytes results in the development of murine hepatocellular carcinomas (HCCs), thus showing that senescence surveillance is important for tumour suppression in vivo. In accordance with these observations, ras-specific Th1 lymphocytes could be detected in mice, in which oncogene-induced senescence had been triggered by hepatic expression of NrasG12V. We also found that CD4+ T cells require monocytes/macrophages to execute the clearance of senescent hepatocytes. Our study indicates that senescence surveillance represents an important extrinsic component of the senescence anti-tumour barrier, and illustrates how the cellular senescence program is involved in tumour immune surveillance by mounting specific immune responses against antigens expressed in pre-malignant senescent cells.

Link: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10599.html

Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders

2011-11-03T17:43:00.003Z

A paper worth reading. Excellent work.

ABSTRACT

Darren J. Baker, Tobias Wijshake, Tamar Tchkonia, Nathan K. LeBrasseur, Bennett G. Childs, Bart van de Sluis, James L. Kirkland & Jan M. van Deursen

Advanced age is the main risk factor for most chronic diseases and functional deficits in humans, but the fundamental mechanisms that drive ageing remain largely unknown, impeding the development of interventions that might delay or prevent age-related disorders and maximize healthy lifespan. Cellular senescence, which halts the proliferation of damaged or dysfunctional cells, is an important mechanism to constrain the malignant progression of tumour cells1, 2. Senescent cells accumulate in various tissues and organs with ageing3 and have been hypothesized to disrupt tissue structure and function because of the components they secrete4, 5. However, whether senescent cells are causally implicated in age-related dysfunction and whether their removal is beneficial has remained unknown. To address these fundamental questions, we made use of a biomarker for senescence, p16Ink4a, to design a novel transgene, INK-ATTAC, for inducible elimination of p16Ink4a-positive senescent cells upon administration of a drug. Here we show that in the BubR1 progeroid mouse background, INK-ATTAC removes p16Ink4a-positive senescent cells upon drug treatment. In tissues—such as adipose tissue, skeletal muscle and eye—in which p16Ink4a contributes to the acquisition of age-related pathologies, life-long removal of p16Ink4a-expressing cells delayed onset of these phenotypes. Furthermore, late-life clearance attenuated progression of already established age-related disorders. These data indicate that cellular senescence is causally implicated in generating age-related phenotypes and that removal of senescent cells can prevent or delay tissue dysfunction and extend healthspan.

Link: http://www.nature.com/nature/journal/vaop/ncurrent/full/nature10600.html

Cellular Senescence and COPD

2011-10-06T20:55:00.012+01:00

Chronic obstructive pulmonary disease (COPD) is characterized by progressively reduced airflow within the lungs, making it difficult to breath. During normal breathing, air sacs (alveoli) (which are elastic) fill up with air and oxygen passes through the air sac walls into the blood. With COPD the air sacs can lose elasticity, the walls between air sacs are destroyed, the walls become thick and inflamed and the airways make more mucus than normal leading to clogging. All these changes contribute to reduced airflow in COPD.

COPD is predominately associated with tobacco smoking and previous studies investigating the pathophysiology of emphysema have demonstrated that cigarette smoke can cause cells to enter cellular senescence (Tsuji et al, 2004, Nyunoya et al 2006). As such, a number of studies have investigated the role of cellular senescence in the development and progression of COPD. Cigarette smoke may trigger cells to senesce directly due to DNA damage or indirectly (if apoptosis is occurring) through increasing cell turnover leading to accelerated telomere shortening.

Senescent cells secrete pro-inflammatory cytokines, growth factors and proteases (most likely for immune clearance) that can cause tissue damage, leading to loss of function of the tissue in which they reside. In the case off COPD the secretion of proteases by senescent cells could result in loss of elasticity of air sacs and destruction of air sac walls. The secretion of cytokines and chemokines by senescent cells would lead to persistent inflammation.

Tsuji et al (2009) has shown that lung tissue of COPD patients contained higher percentages of senescent alveolar cells displaying a pro-inflammatory phenotype compared with tissue from asymptomatic smokers and non-smokers. Noureddine et al (2011) has demonstrated that pulmonary artery smooth muscle cell (PA-SMC) senescence is an important contributor in the process of pulmonary vessel remodeling in COPD patients. Senescent PA-SMC were shown to stimulate cell growth and migration of normal PA-SMC through the release of paracrine soluble and insoluble factors. Dogauassat et al (2011) have shown that lung fibroblasts in smokers and ex-smokers with moderate COPD display a senescent phenotype. This study suggests that even after stopping smoking, the persistence of senescent cells may still contribute to COPD. Amsellem et al (2011) have recently showed that premature senescence in pulmonary vascular endothelial cells may contribute to inflammation in COPD.

Research also suggests that patients with COPD have a two to six times more chance of developing lung cancer compared with people of normal lung function (COPD Foundation). It could be speculated that the presence of senescent cells in COPD patients may increase the chances of lung cancer. It has been shown that the secretory phenotype of senescent cells can play a role in cancer development by stimulating growth and angiogenic activity of pre-malignant cells (reviewed in Campisi and d'Adda di Fagagna, 2007). Additionally, stochastic epigenetic/genetic alterations within senescent cells may allow them to escape the senescence growth arrest, thus becoming cancerous.

Publications

Tsuji T, Aoshiba K, Nagai A. Alveolar cell senescence exacerbates pulmonary inflammation in patients with chronic obstructive pulmonary disease. Respiration. 2010;80(1):59-70. Epub 2009 Dec 17.

Noureddine H, Gary-Bobo G, Alifano M, Marcos E, Saker M, Vienney N, Amsellem V, Maitre B, Chaouat A, Chouaid C, Dubois-Rande JL, Damotte D, Adnot S. Pulmonary artery smooth muscle cell senescence is a pathogenic mechanism for pulmonary hypertension in chronic lung disease. Circ Res. 2011 Aug 19;109(5):543-53. Epub 2011 Jun 30.

Amsellem V, Gary-Bobo G, Marcos E, Maitre B, Chaar V, Validire P, Stern JB, Noureddine H, Sapin E, Rideau D, Hue S, Le Corvoisier P, Le Gouvello S, Dubois-Randé JL, Boczkowski J, Adnot S. Telomere Dysfunction Causes Sustained Inflammation in Chronic Obstructive Pulmonary Disease. Am J Respir Crit Care Med. 2011 Sep 8. [Epub ahead of print]

immune clearance of senescent and cancer cells

2011-09-30T22:53:00.012+01:00

The age-associated increase in the incidence of disease development and cancer occurrence is often thought to be due to the gradual accumulation of damage over the lifetime of an organism. However, an alternative opinion is that damaged cells are effectively eliminated and replaced by the immune system and regenerative cells (stem cells) and only when this “remove and replace” system failures, do organisms begin to show signs of ageing.

The presence of persistent DNA damage triggers cells to enter senescence (irreversible growth arrest) to protect the cell from becoming cancerous. The presence of the persistent DNA damage in these growth-arrested cells appears to activate pathways leading to cytokine/chemokine secretion and presentation of cell surface ligands (i.e MICA, MICB, ULBP2) which can be recognized by natural killer cells (NK) and some T-cells. This may allow damaged/senescent cells to communicate with immune cells for their removal (although more evidence of this is required).

For cells to become cancerous, they need to bypass senescence (following irreparable DNA damage), often achieved by acquiring mutations in genes associated with activation and maintenance of the senescence growth arrest. When such cells bypass senescence, the persistence of DNA damage may also activate pathways leading to cytokine/chemokine secretion and presentation of NK ligands.

It is also possible that cells can become cancerous if they instead escape senescence. “Escaping” is different from “bypassing” in that these cells were once senescent. If senescent cells persist in tissues without immune clearance, it is possible that stochastic genetic/epigenetic changes may lead to activation/inactivation of genes that allow the once senescent cell to reneter the cell cycle. A consequence of this escape may be the maintenance of the pro-survival phenotype and the pro-inflammatory phenotype associated with senescence. Escaping senescence may be more pertinent in cancer cells that have become senescent in response to therapy. Escape from senescence in this instance may lead to the progression of more aggressive cancers.

I am not aware of any studies that have investigated the similarities/differences in the secretory phenotype/NK ligand activation of senescent verses cancer cells. However, if both exist due the DNA damage response activating the immune response (DDR-AIR), then they are probably very similar.

If senescent and cancer cells were always effectively being removed then the incidence of cancer and disease would greatly be reduced. However, age-associated cancer and disease does occur and this may in part be due to a failure in the immune system to effectively remove senescent/cancer cells as we age. Additionally, cancer cells can develop various strategies for evading the immune response (i.e secretion of immunosuppressive cytokines). Whether the same strategies occur in senescent cells remains to be discovered.

Although purely speculative, it is possible that some of these strategies for evading immune surveillance is a result of pro-longed exposure to the pro-inflammatory phenotype of these cells. There may be a limited biological time frame whereby the presence of the inflammatory phenotype is beneficial for cell removal. Longer exposure may lead to an adaptive response through autocrine signalling leading to changes that evade immune surveillance. For example, the secretion of immunosuppressive cytokines may be an adaptive response for preventing detrimental damage from long exposure to pro-inflammatory cytokines.

Ongoing and future investigations should aim to provide solid evidence of whether (1) the secretory phenotype of senescent cells is for the purpose of immune clearance, (2) and if so, does immune clearance fail or become impaired with age and (3) if it does fail, what are the mechanisms?

Stem Cell Backup: Bank your cells for the future

2011-04-13T19:18:00.003+01:00

The following is a brief article provided by Stem Cell Backup (click here), which discusses the importance of banking your cells for future therapeutic applications.

Growing replacement ears for injured soldiers. Allowing the paralyzed to walk again. Restoring sight to the blind. Curing multiple sclerosis. Growing transplantable lungs. This, and more, is being done today. The magic technology? The most basic there is, the patient's own cells. You are witnessing the dawn of a new era of medicine. Regenerative medicine—using your own stem cells to heal yourself—is no longer science fiction. The U.S. Department of Health and Human Services reports that “regenerative medicine is the vanguard of 21st century health care.” These experts estimate that half of Americans now under the age of 65 will receive regenerative therapies during their lifetime. Simultaneously, groundbreaking advances now mean scientists can use your own non-stem cells to make the stem cells used in regenerative medicine.

In 2006 a Japanese researcher did something that most researchers considered impossible, he 'reprogrammed' a normal skin cell and made it into a stem cell. The new technique was so effective and technically simple that thousands of research laboratories soon began using these 'induced' pluripotent stem cells (iPSC). Even the scientist who cloned Dolly the sheep abandoned cloning, saying "[Reprogramming is] 100 times more interesting [than cloning]…I have no doubt that in the long term, direct reprogramming will be more productive” (London Telegraph 11/10/08).

High expectations, to be sure, but iPSC are already exceeding them. Researchers have treated or even fully cured maladies like Parkinson's, heart attack damage and diabetes in test animals using iPSC. Additionally, iPSC have also been used to grow dozens of types of transplantable tissue, like retinas, and even fully-functioning organs, like livers. Medical experts expect iPSC to play a role in virtually every medical treatment of the future.

To help you take advantage of these dual advances, Stem Cell Backup banks your cells for your future use. Like many things in life, age matters. Research shows that cells taken from older patients are less effective for therapeutic use. By banking your own youngest, healthiest cells you can grow any kind of new tissue you need, whether heart, liver, or muscle.

"You are seeing the birth of a new industry," says Patrick O'Malley, president of Stem Cell Backup, "that has a strong precedent in the long-established cord blood industry. In the U.S. alone, over one million families currently bank their newborn child’s umbilical cord blood for future medical treatments. Banking your own cells is like cord blood for the rest of us." Mr. O’Malley points to the universal consensus of medical experts who expect great things from this new form of personalized medicine, "Every knowledgeable expert says that this technology is transformational. The Nobel Laureate for Medicine said, 'This is going to be the way forward. …We’ve all been waiting for this' (WSJ 11/21/07). Doctor Oz predicted on Oprah that a patient’s own cells will be used to cure Parkinson's disease in 8 or 9 years."

Stem Cell Backup was founded in 2008 to allow individuals to take advantage of new discoveries in stem cell medicine. After extensive research and testing, the company began accepting client samples in 2011. Stem Cell Backup is the first and only company to allow individuals to easily, safely, and inexpensively save their own cells for use in future medical therapies. It has a processing laboratory in the U.S. and is currently identifying local partner candidates in European and Asian markets.

For those who want to take advantage of this unique service, visit http://www.stemcellbackup.com/ for further details.

23andme: Using Personal Genome Analysis (PGA) for Promoting Healthier Longer Lives

2011-03-22T01:02:00.013Z

I recently got my genome analysis results back from 23andme. 23andme analyse your genome for single nucleotide polymorphisms (SNPs), variations in single nucleotides, which can correlate with disease, drug response and other phenotypes.

Personal genome analysis (PGA) will revolutionize how we think about our own health. Currently, whenever a symptom or illness presents itself, we see doctors and those doctors provide medicines and treatments to remove or control the problem. With PGA we can discover what diseases we may be at risk of developing and in some cases adjust our lifestyles to reduce/prevent disease occurrence/progression.

For example, my PGA suggests (based on a number of studies) that I am at risk of developing a mild form of hemochromatosis, a disorder in which the body absorbs too much iron, causing damage to tissues, specifically the liver. A fact I find rather amusing considering I nearly started a research project focused on the role of iron in ageing.

Knowing I may be at risk of hemochromatosis, I can alter my diet to avoid foods such as red meats, which have a high percentage of iron. Thus, having this genetic knowledge could postpone/prevent the appearance of a disease by taking the appropriate steps. Prevention of disease, means healthier, longer lives.

There have also been a few studies looking at SNP’s associated with longevity. One study compared 213 Ashkenazi Jewish subjects ranging in age from 95 to 107 to a group of counterparts about 30 years their junior. Members of the longer-lived group were more likely to have a C in both copies of the SNP rs2542052. People with this genetic signature tended to be more sensitive to insulin and were less likely to have high blood pressure, which suggests it may promote longevity by protecting against cardiovascular disease. Luckily for me, my PGA is telling me I have two copies C.

Another study, which is more related to longevity in the Asian population, compared 213 Japanese men who lived 95 years or longer to 402 Japanese men who died before the age of 81. The researchers found that the longer-lived Japanese men were more likely to have a C at one or both copies of rs2764264, a SNP in the FOXO3A gene. Each C at rs2764264 was associated with about 1.6 greater odds of reaching age 95 or beyond compared to having a T at this position. The FOXO3A gene has been shown to modulate longevity in laboratory animals. Again, I have two copies of C (although in this instance, it may be ethnicity specific).

Readers must take into account that these studies were based on a limited number of participants and environmental factors such as diet are probably just as, if not more important than the underlying genetics. But that is a debate for another time.

Unfortunately it may not all be good news, I found out I have an increased chance of developing male pattern baldness…..thanks Dad :-)

www.23andme.com

If you are someone who have either bought a genetic test from a company like 23andme, or are thinking of doing so, please help out Corin Egglestone, a PhD student from Loughborough University in the UK, by spending 10 minutes of your time to answer questions for her survey: http://www-staff.lboro.ac.uk/~lsctre3/survey.html

Novelli V et al. (2008) . “Lack of replication of genetic associations with human longevity.” Biogerontology 9(2):85-92.

Atzmon G et al. (2006) . “Lipoprotein genotype and conserved pathway for exceptional longevity in humans.” PLoS Biol 4(4):e113.

Willcox BJ et al. (2008) . “FOXO3A genotype is strongly associated with human longevity.” Proc Natl Acad Sci U S A 105(37):13987-92.

Guest Blog: Harold Katcher: Is Prevention of Ageing within our Grasp?

2011-03-01T03:05:00.003Z

Introduction

Slowly but steadily knowledge about the human body has progressed and new ideas of animal ageing have immerged. The classic model of ageing, based on “accumulation of errors” has become an outdated notion. Instead, evidence suggests that ageing, at least in part, is likely the result of a failure in the function of cells (such as stem cells) required for cellular regeneration. Replacing impaired stem cells with fully functional stem cells should thus prevent/treat age-associated pathologies allowing us to live healthier longer lives.

What We Think We Know

We were once taught that the essential differences between animals and plants were that plants are mostly non-living, except for a layer or bud of special cambium cells, called meristems; unlike animals, plants grew from their outside surfaces and tips – while animals grew from the inside by the division of somatic cells. These notions have since been replaced with one in which specialized cells, called stem cells, (the animals' “meristematic” tissue) or progenitor cells (like stem cells, only less pleuripotent and of a limited lifespan), that can differentiate into, and replace, various diverse cell-types, (in contrast to somatic cells which cannot). (Janzen et al 2006). It has become clear that the many impairments of the ageing body are due to ageing stem/progenitor cell populations.

For example, muscle loss in the elderly (sarcopenia) appears to be the result of decreasing numbers of stem cells. (Hawke T.J..& Garry, D.J-. 2001). Muscle satellite cells which lie between the sarcolemma and the basement membrane of terminally differentiated muscle fibers, provide muscle precursor cells that are then incorporated into muscle fibers (Mauro, A . 1961). Satellite cells from aged individuals display an impaired proliferative ability when compared with satellite cells from a young individual, thus possibly resulting in sarcopenia. In organs like the liver that depend on progenitor cells for tissue repair and replacement, progenitor cell impairment would also result in deficiencies in wound healing and thus presentation of age-associated pathologies.

The loss of immune function, commonly observed within the elderly, makes them more susceptible to various diseases, infections and cancers. As in the case of ageing tissues, a lack of functional cells characterizes an aged immune system. Conversely, in this instance, stem cell populations do not decline, but instead there is an increase in the stem cell populations (i.e. hematopoietic stem cells, HSCs) that reside within the bone marrow (Sudo et al. 2000). However, unlike young HSCs, the ratio of the many potential cell-types that the HSC population generates changes with ageing. For example, aged HSCs move away from production of lymphoid line cells (T and B lymphocytes and NK cells) and towards the production of myeloid line cells (monocyte/macrophages, RBC, thrombocytes, granulocytes) cells. This age-associated reduction lymphoid cells, which forms the adaptive immune system, is thought to result in the age-associated decreased immune response (Chambers et al. 2007). The increased fraction of myeloid precursor HSCs appears to contribute to the myeloid leukemias that occur among the elderly (Rossi et al.).

So how can we combat the effects of functionally declining stem/progenitor stem cell populations? Solutions such as stem cell cloning and telomere elongation through telomerase therapy have been suggested, but is this really necessary? Is there a way to rejuvenate aged stem cells from within out own bodies, giving them the ability to constantly maintain high cell numbers in the organs they populate, cells with high proliferative capacity, rapid responses to wounding? It has become apparent that this possibility may exist.

A New Paradigm - Evidence accumulates

Several line of evidence suggest that the standard model of ageing, based on “error accumulation” is incorrect. Several studies in which tissues or organs are transplanted from donor animals have shown that the ability of the graft to be successful (by measures of ability to proliferate or recover from wounds) depends not on the donor's age, but on the age of the recipient. Such studies have shown that HSCs from aged immunodeficient donors gave normal responses in young recipients (Harrison et al 1977), and that aged HSCs could be coaxed to produce lymphoid cells by being placed together with young osteolineage cells (Mayack, S,R. And Wagers, A. 2008). Additionally, transplanted aged muscle responded to the internal environment of a young recipient by showing the same sort of wound- repair as young muscle.

The most important experiment investigating the effect of environment (specifically the humoral environment) was performed in 2007 by Irina Conboy and a later confirmation came with experiments performed by Mayack's group in 2010. While earlier in vivo experiments showed that tissues and organs obtained from aged donors could effectively be rejuvenated by being placed in the bodies of young recipients, it was not clear which factors were acting to rejuvenate these aged organs. Were there local tissue interactions, were there positive factors in young recipients that caused a revitalization of the old organs, or perhaps negative factors in aged bodies preventing cells from proliferating? Were cells from the young recipients colonizing these aged organs? How much did the environment of the aged cells influence their phenotype?

Conboy et al (2005) used a procedure called parabiosis (Finerty, J. 1952) to pair the circulatory systems of two mice. They now effectively shared the same blood, but not interactions between tissues of the parabionts (other than blood cells), thereby narrowing down the possible factors influencing the cells of the parabionts. In a nicely controlled experiment, mice were paired in either isochronic parabiosis or heterochronic parabiotic associations – in the isochronic cases two mice of the same age were tied together – either a young-young pairing or an old-old pairing and the heterochronous association a young mouse (2-3 months) was coupled to an old mouse (19-26 months) and were kept in this pairing for five weeks. After that time, it was found that in heterochronic pairings, but not in isochronic pairings old muscle satellite cells returned to youthful performance in terms of effecting wound healing and increased proliferative capacity. Another insightful experiment narrowed the range of responsible factors. In vitro experiments showed that exposure of aged cells to young serum was sufficient to rejuvenate aged HSCs, muscle satellite stem cells as well as liver progenitor cells. As the paired mice parabionts have distinctive chromosomal markers it was assured that the old organs weren't being colonized by young cells.

Further experiments extending the concept that the environment controlled the age-phenotype of the cell, was provided by Mayack et al (2010). Mayack used Conboy's method of parabiosis together with parallel in vitro studies using serum to provide the external environment. Both sets of experiments also showed that young serum was caple of rejuvenating aged HSCs. Mayack's group however showed that the cells rejuvenated by the young environment were the bone stromal cells. It was these rejuvenated stromal cells that later interacted with aged HSCs to set back their phenotypic-age. The parallel in vivo/in vitro experiments performed by these groups showed that the rejuvenation of cells was a function of a factor or factors carried in the serum. The explanations proposed; that either young blood diluted inhibitory factors present in aged blood, or brought new levels of stimulatory factors carried by young blood, or both.

While neither experiment could discriminate between these alternatives, both showed that the cells' environment was responsible for an ageing-phenotype (the panoply of genes expressed, its proliferative potential, various molecular markers of ageing). The one conclusion that can be taken for certain is that factors in the blood of the young animal were able to rejuvenate a variety of different stem/ progenitor cell lines in vivo, and that, in particular, as show by the in vitro experiments, factors present in the serum of young animals rejuvenate the stem and progenitor cells of aged animals. The conclusion reached by the groups involved in this research was that blood borne determinants, both positive and negative might be isolated, and eventually added to or removed from the blood of the ageing. So for the first time in history, there is a reasonable prospect to achieve what mankind has sought for all of history. There may finally be a therapeutic approach for the treatment of ageing and thus, disease. Evidence of such inhibitory factors in the blood of aged mice (McCay et al. 1957) and stimulatory factors in the serum of young mice (Hadad et al 1988), have already been detected.

Conclusions

The answers to extending healthy life span is now within our grasp – what if our own stem cells could be rejuvenated? With only the four cell types proven to be “rejuvenate-able”, (1) muscle loss could be eliminated, (2) the immune system made effective again, (3) bone now capable of making osteoblasts for growth and strength and (4) the liver able to perform its functions as in youth. Other cell types may also be positively influenced, leading to youthful changes such as, new hair growth, smooth skin, improved memory from neuron regeneration. The possibilities are endless. If viewed in this light, it is obvious what should be done – this new model should be tested and tested on people – and the means to test it? A practical medically approved procedure, cheap while being at the same time, able to provide all of the factors needed to rejuvenate cells is available right now! This is a procedure that any consenting physician could perform tomorrow.

I am not going to talk about it now – like all great secrets, once told it becomes obvious –“ no duh, why hasn't it already been tried.” Join me and we'll perhaps try it together. (hkatcher@earthlink.net.)

Papers of Interest

Chambers, S.M. et al. Ageing hematopoietic decline in function and exhibit epigenetic dysregulation. PLOS Biology (5) e201

Conboy et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment Nature (433) 760 -764 (2005)

Finerty, J. Parabiosis in physiological studies Physiol. Rev. (32) 277 – 302 (1952)

Hadad, E. J. et al Lymphocyte induced angiogenesis factor is produced by L3T4 murine lymphocytes and its production declines with age. Cancer Immunol Immunother (26) 31- 34 (1988)

Harrison et. al. Stem cell lines from an old immunodeficient donors give normal response in young J. Immunology (118)1223 – 1228 (1977)

Hawke, T.J. And Garry, D.J. Myogenic satellite cells: physiology to molecular biology J. Appl. Physiol (91) 534 – 551 (2001)

Janzen, V. et al. Stem-cell ageing modified by the cyclin-dependant kinase inhibit p12INK4a . Nature (443) 421- 426 (2006)

Mauro, A. Satellite cell of skeletal muscle fibers. J. Biolphys.Biochem. Cytol (9) 493 – 495 (1961)

McCay et al. Parabiosis between young and old rats Gerontologia; (1):7-17 ( 1957)

Mayack, S.R. et al. Systemic signals regulate ageing and rejuvenation of blood stem cell niches Nature (463)495-500 (2010)

Mayack, S.R. & Wagers, A Osteolineage niche cells initiate hematopoietic stem cell mobilization Blood (112) 519 – 532 (2008)

Rossi, D.G., Jamieson, C.H. & Weissman, I.L. Stem cells and the pathways to ageing and cancer. Cell (132) 681-696 (2008)

Sudo, K. et al. Age-associated characteristics of murine hematopoietic stem cells. J. Exp. Med (193) 1273 – 1280 (2000)

The Science of Ageing – Global Progress

2010-11-23T19:40:00.002Z

Prof Richard Faragher on the science of ageing

2010-08-05T22:12:00.002+01:00

Taken from the Science Network: http://thesciencenetwork.org/

Cell-type exclusive senescent phenotype (CESP)

2010-07-28T23:09:00.006+01:00

When cells become senescent, they often display a senescence-associated secretory phenotype or SASP, which consists of cytokines, growth factors and proteases (Coppé et al, 2008). The SASP appears to be similar to a wound healing response and so may function in senescent cell removal. However, if senescent cells persist in tissues (possibly due to an impairment in the removal process) then the local tissue is constantly exposed to the SASP. The secretion of these senescence-associated factors has the potential to detrimentally alter the local microenvironment, leading to tissue dysfunction associated with ageing and disease.

In addition to the SASP there also appears to be a cell-type (or even cell-strain) exclusive senescent phenotype or CESP, an area of research underexplored. Microarray data of senescent cells show a differential expression of genes unrelated to SASP, which is nevertheless cell-type/strain specific (Shelton et al, 1999, Zhang et al, 2003, Burton et al 2009b, unpublished data). These transcriptional changes likely do not perform a specific physiological function (unlike the potential wound healing response of the SASP) and may be caused by random changes associated with alterations to the chromatin structure during senescence (Zhang et al, 2003, Funayama and Ishikawa, 2007). However, these altered transcriptional profiles may manifest into changes associated with altered morphology, behaviour and function and such changes could have a detrimental impact on tissue. Additionally, components of the SASP may directly influence changes associated with a CESP by activating pathways through autocrine/paracrine signaling. Different cell-types may respond differently to the SASP and this may lead to a CESP.

Examples of CESP are currently limited because the majority of work involving senescence has been carried out on fibroblasts with less emphasis on cell-types linked to disease development.

One example of a CESP is the decrease in nitric oxide synthase (NOS) activity observed in senescent vascular endothelial cells (Matsushita et al. 2001, Minamino et al. 2002). The synthesis of nitric oxide (NO) by NOS in vascular endothelial cells is important for maintaining vascular homeostasis and a decrease in NO has been suggested to be a potential risk factor in cardiovascular disease (Cannon, 1998).

Another example of a CESP is the insufficient insulin release observed in senescent pancreatic beta cells (Sone and Kagawa, 2005). Due to the importance of insulin release in regulating glucose metabolism, an impairment in this function is associated with the pathogenesis of type II diabetes.

A CESP associated with senescent vascular smooth muscle cells (VSMCs) appears to be the shift to a pro-calcificatory/osteoblastic phenotype (Burton et al, 2009, Nakano-Kurimoto et al, 2009). VSMCs play an important role in the contraction and relaxation of blood vessels, a mechanism that is responsible for the redistribution of the blood within the body to areas where it is needed. The pro-calcificatory phenotype of senescent VSMCs could thus impair this function and this could contribute to cardiovascular dysfunction.

To further understand the mechanisms associated with age-related tissue dysfunction and disease development, research should focus not just on the impact of the SASP but also the CESP.

Papers:

Matsushita H, Chang E, Glassford AJ, Cooke JP, Chiu CP, Tsao PS (2001) eNOS activity is reduced in senescent human endothelial cells: Preservation by hTERT immortalization. Circ Res 89:793–798

Sone H, Kagawa Y. (2005) Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice. Diabetologia. Jan;48(1):58-67. Epub 2004 Dec 29.

Burton DG, Matsubara H, Ikeda K. (2010) Pathophysiology of vascular calcification: Pivotal role of cellular senescence in vascular smooth muscle cells. Exp Gerontol. 2010 Jul 18. [Epub ahead of print]

VSMC senescence and calcification

2009-10-08T17:04:00.014+01:00

A paper by Japanese researchers has recently been published (click here) demonstrating the importance of vascular smooth muscle cell (VSMC) senescence in calcification. Vascular calcification is important because it can lead to reduced elasticity and compliance of arteries and is also a prominent feature of advanced atherosclerotic plaques. Although the process of calcification appears to be similar to that of bone formation (Abedin et al, 2004), little is known about the underlying mechanism. Nakano-Kurimoto et al have confirmed some of the findings published earlier by Burton et al (click here), but have taken it many steps forward with their in-depth investigation.

As well as providing further evidence for a role of VSMC senescence in calcification, these studies also demonstrate the importance of understanding the senescent specific changes which may occur in cells associated with age-related disease/dysfunction. Such an understanding may not only provide answers regarding mechanisms of disease development, but may also provide biomarkers of tissue specific ageing.

Replicative senescence of vascular smooth muscle cells

enhances the calcification through initiating the osteoblastic

transition

Nakano-Kurimoto R, Ikeda K, Uraoka M, Nakagawa Y, Yutaka K, Koide M, Takahashi T, Matoba S, Yamada H,Okigaki M, Matsubara H

Medial artery calcification, which does not accompany lipid or cholesterol deposit, preferentially occurs in elderly population, but its underlying mechanisms remain unclear. Here, we investigated the potential role of senescent vascular smooth muscle cells (VSMCs) in the formation of senescence-associated medial calcification. Replicative senescence was induced by the extended passages (until passage 11-13) in human primary VSMCs, and cells in early passage (passage 6) were used as control young cells. VSMC calcification was markedly enhanced in the senescent cells comparing with that in the control young cells. We identified that genes highly expressed in osteoblasts, such as alkaline phosphatase (ALP) and type-I collagen, were significantly up-regulated in the senescent VSMCs, suggesting their osteoblastic transition during the senescence. Knockdown of either ALP or type-I collagen significantly reduced the calcification in the senescent VSMCs. Of note, runt-related transcription factor-2 (RUNX-2), a core transcriptional factor that initiates the osteobalstic differentiation, was also up-regulated in the senescent VSMCs. Knockdown of RUNX-2 significantly reduced the ALP expression and calcification in the senescent VSMCs, suggesting that RUNX-2 is involved in the senescence-mediated osteoblastic transition. Furthermore, immunohistochemistry of aorta from klotho-/- aging model mouse demonstrated in vivo emergence of osteoblast-like cells expressing RUNX-2 exclusively in the calcified media. We also found that statin and Rho-kinase inhibitor effectively reduced the VSMC calcification by inhibiting Pi-induced apoptosis and potentially enhancing matrix Gla protein expression in the senescent VSMCs. These findings strongly suggest an important role of senescent VSMCs in the pathophysiology of senescence-associated medial calcification, and the inhibition of osteoblastic transition could be a new therapeutic approach for the prevention of senescence-associated medial calcification. Key words: vascular calcification, medial calcification, senescence, RUNX-2.

Also see: DISEASE FOCUS: Atherosclerosis and vascular calcification

The removal of senescent cells using therapeutic agents

2009-06-29T17:52:00.003+01:00

As discussed in the previous blog, one of the strategies for overcoming the detrimental effects of senescent cells is to remove them as they appear through the use of therapeutic agents. At present, no drug-based system exists which can specifically identify senescent cells and remove them. However, there is currently great interest in the development of drugs which specifically target and remove cancer cells. The problem with current cancer treatments (such as drugs used in chemotherapy) is that they are non-specific and as such can cause damage and undesirable changes to non-cancerous cells, causing side-effects. The development of cell-specific drug targeting is greatly needed and such research could be adapted to target senescent cells. Cell-specific drug targeting requires a carrier molecule containing a targeting agent which specifically recognises and binds to a specific receptor or binding site on the surface membrane of target cells and a therapeutic agent which could trigger programmed cell death, apoptosis. The following are crucial factors in determining the success of drug-targeting systems (Beljaars et al, 2001, Petrak 2005).

(1) Cellular specificity: For a drug to exert its desired effect it needs to be in physical contact with its physiological target, such as a receptor.

(2) Rate of elimination of the drug-carrier conjugate: It is essential that the drug-carrier conjugate is not removed too rapidly from the circulation. If it is eliminated from systemic circulation more rapidly than it is delivered to the target site, the amount of conjugate at the target site might never be enough to provide the required concentration of free (unbound) drug.

(3) Rate of release of free drug at the non-target site: Depending on the amount of drug, the release of drug away from the target site could nullify any benefits that might potentially come from delivering the drug to the target site.

(4) Rate of delivery of drug-carrier conjugate to the target site: If the drug conjugate reaches the target site too slowly, the supply of free drug might never be sufficient to generate the concentration required to elicit the desired therapeutic effect at the site of action.

(5) Rate of release of free drug at target site: The capacity of the system selected for the release of free drug from the conjugate should be considered. It needs to be suitable for processing the entirety of the drug-carrier conjugate arriving at the target site, doing so at a rate that also ensures drug accumulation at this site.

(6) Rate of removal of free drug from the target site: Drugs that benefit most from target-selective delivery are those that are retained at the site while acting on their target of action.

(7) Rate of elimination of the drug-carrier conjugate and free drug from the body: For optimal targeting, elimination of the complete drug-carrier system should be minimal.

One promising area of research in the development of drug delivery systems incorporates the use of nanotechnology (http://nano.cancer.gov/). Such technology has been used to create dendrimers, spheroid or globular nanostructures which are highly branched (Alexis et al, 2008). The branched regions of these dendrimers can be used to attach molecules such as targeting and therapeutic agents (Gillies and Frechet 2005). To test this nano-delivery system, invesitgators at the University of Michigan attached a targeting agent, a therapeutic agent and an imaging agent to the surface of dendrimers (Majoros et al, 2006, Shi et al 2007). The investigators chose folic acid as the tumour-targeting agent (a molecule which binds to a high-affinity receptor found on many types of tumour cells), paclitaxel as the therapeutic agent (a drug which triggers programmed cell death, apoptosis) and the fluorescent dye known as fluorescein isothiocyanate as the imaging agent. This nano-dilivery system was then tested on two sets of cancer cells in vitro: one that expresses the folic acid receptor and one that does not. Only the cells containing the folic acid receptor took up the dendrimer, visualised by the presence of the imaging agent. The dendrimer construct was highly toxic to these cells but had no effect on cells without the folic acid receptor. When both of these cells were exposed to dendrimers containing the targeting and imaging agent but no paclitaxel, no detrimental effects were observed.

These promising initial results thus call for tests to be carried out on animals with tumours that overexpress folic acid receptors. It is research like this that could one day be adapted to specifically target senescent cells. For this to be the case, a target agent is required that specifically recognises senescent cells. For this to be achieved, a deeper understanding of the changes which occur when a cell becomes senescent is required. Ideally a universally expressed senescent membrane receptor would be ideal, but at present no such receptor is known. If it did, it would also make a useful biomarker for detecting senescent cells in tissues.

Cellular Senescence in Anti-Ageing Research

2009-06-29T17:28:00.003+01:00

Introduction

The accumulation of senescent cells (cells which have undergone permanent growth arrest) in tissues is thought to contribute to the development/progression of age-related disease and disability. Why? Partly because when cells become senescent, their gene expression becomes radically altered and as a result secrete proteins that damages the body. Growth-competent cells can become senescent as a result of telomere shortening. Telomeres are a region of repetitive DNA at the end of chromosomes, important in chromosome stability. Every time a cell divides, telomeres gradually become shorter and shorter until they trigger a response which causes them to enter senescence. This is known as replicative senescence. However, an enzyme known as telomerase can lengthen telomeres and thus prevent a cell from becoming senescent.

Telomerase is an enzyme which consists of an RNA molecule and a catalytic component known as hTERT. It is a reverse transcriptase which uses its RNA component as a template to reverse transcribes DNA back to the ends of chromosomes. Telomerase activity is repressed in most somatic cells and reactivated in ~90% of human cancers (Artandi, 2006). Introduction of telomerase into normal somatic cells has been shown to extend replicative life-span (Bodnar et al, 1998) and not induce changes associated with a malignant phenotype (Jiang et al, 1999).

Since senescent cells are potentially detrimental to the tissues in which they reside, anti-ageing research has three main aims for dealing with this problem:

(1) Prevention: prevent cells from becoming senescent.

(2) Removal: remove senescent cells as they appear.

(3) Replacement: replacement of cells which have naturally or artificially been removed.

PREVENTION: Telomerase Therapy

Telomerase therapy is aimed at preventing the appearance of senescent cells in tissues by lengthening telomeres in somatic cells. At present, this is not possible. It is possible to get cells to express telomerase in culture by insertion of the hTERT gene (Bodnar et al, 1998), but there is currently no technology which can insert the hTERT gene into every cell in the body. Since every cell in the body already has the gene for hTERT (it is just not activated) a better alternative approach is the development of drugs which “turn on” the hTERT gene. This is the main focus for companies like Sierra Sciences.

Problems associated with Telomerase Therapy

Apart from the problem of turning on telomerase expression in all the cells of the body, there are a number of other issues that need to be questioned.

(1) Not all cells enter senescence as a result of telomeres shortening: Some cell types, such as keratinocytes (Darbro and Klingelhutz, 2004), and possibly astrocytes and corneal endothelial cells (unpublished) enter senescence by a mechanism independent of telomere shortening. As such, cellular senescence cannot be prevented by the addition of telomerase.

(2) Cellular senescence can be triggered as a response to DNA damage: Even if telomeres are elongated, cells can still become senescent as a result of DNA damage. It is not known what fraction of senescent cells in tissues is due to replicative senescence or the result of DNA damage.

(3) Cancer risk: The risk of cancer is likely to be great if telomerase is constantly being expressed in cells, but if telomerase expression is transiently expressed by drugs then this risk would be minimised.

REMOVAL: Therapeutic agents and/or the use of the Immune System.

All three of the above problems associated with telomerase therapy could be eliminated if senescent cells were removed as they appeared in tissues. Prevention therapies should therefore be applied along side removal strategies. Two possible approaches for removing senescent cells are:

(1) The use of therapeutic agents (drugs) to specifically target and destroy senescent cells.

(2) The use of our own immune system to remove senescent cells.

Use of Therapeutic Agents

Therapeutic agents have the potential to specifically target senescent cells and induce programmed cell death (apoptosis). At present, no such drug is available. However, drugs that are being developed to specifically target cancer cells could one day be adapted to target senescent cells. For this to be made possible, a cell surface marker specific to all senescent cells needs to be identified. A drug can then be developed which specifically identifies that marker, binds to it and induces apoptosis. A more detailed review of cell specific drug targeting will be presented at a later date.

Use of the bodies own immune system

Cancer cells (and possibly senescent cells) may persist in tissues in later life because the immune system fails to remove them (see here). Why? Because the immune system is also governed by ageing mechanisms, and as we age the immune systems ability to remove cancer and senescent cells is gradually impaired. An understanding of the mechanisms which lead to functional decline in the immune system is thus needed for the development of anti-ageing therapies. This is discussed in more detail at a later date.

The use of these two removal strategies without the use of telomerase therapy could be more harmful than good. The removal of one cell only promotes the division of another, thereby reducing the replicative capacity of cells and increasing the appearance of senescent cells. However, if cell removal strategies are used in conjunction with telomerase therapy (at least in some cell types), the negative impact normally observed with cell replacement may not be seen.

REPLACEMENT

If a senescent cell is removed from tissue without the use of telomerase therapy, surrounding cells will divide to replace it, thus decreasing the replicative capacity of those cells and increasing the appearance of senescent cells. Replacement strategies focus on the use of stem cells to replace lost and damaged cells. Stem cells naturally replace lost cells in tissues but it is not known to what extent both stem cells and the surrounding somatic cells play in this process. Also, the functional ability of stem cells has been shown to decline with age in tissues (Sharpless and DePinho, 2007), so the addition of functional stem cells into tissues would be beneficial. Interestingly, it may the the presence of senescent cells that is having a detrimental impact on the functional ability of stem cells. The microenvironment of stem cell niches is important for the normal functioning of these cells (Boyle et al, 2007). Therfore, the presence of senescent cells with their altered secretome may alter the environment of the stem cell niche, thus altering their ability to function properly. The removal of senescent cells alone may therefore partly prevent the age-related decline in stem cell function, providing a stronger repair process.

Conclusion

Like all anti-ageing research, telomerase therapy, senescent cell removal and cell replacement are at their infancy. Only with time, money, a deeper understanding of the ageing process and a motivation to succeed, will we begin to see the inevitable benefits of anti-ageing research.

Burton (2009) cellular senescence, ageing and disease

Replicative lifespan of fibroblasts in ageing studies

2009-06-20T00:12:00.007+01:00

A recent paper by Maier and Westendorp (2009) focused on the replicative capacity of fibroblasts from patients with accelerated ageing syndromes, patients with age-related diseases and donors of varying chronological age. Their findings were as follows:

(1) Fibroblasts from patients with accelerated ageing syndromes are lower when compared with strains from age-matched controls.

(2) No difference in replicative capacity was found in fibroblasts from patients with age-related diseases when compared to age-matched controls.

(3) No relationship between replicative capacity of fibroblasts and donor age.

It is probably not surprising that there is a lower replicative capacity in skin fibroblasts taken from patients with Werner- and Hutchinson-Gilford syndrome patients as the mechanisms underlying these syndromes are probably universally found throughout all the somatic cells in these patients. For example, Werner syndrome is caused by a mutation in the WRN gene and is associated with short telomeres and accelerated cellular senescence (Cox and Faragher, 2007). This mutation is going to be present in all cell types, therefore it does not matter which cell type is investigated, the result of a reduced replicative capacity is likely to be the same. However, the same result is unlikely to be true when investigating the replicative capacity of skin fibroblasts in subjects suffering from diseases associated with a completly different cell type.

Maier and Westendorp investigated the replicative capacity of skin fibroblasts in patients with age-related disease. However, some of the diseases classed as age related in this instance are not. These include cystic fibrosis and familial Alzheimer’s disease. This is not the main point in question. It is not surprising that there is no relationship between the replicative capacity of skin fibroblasts in patients suffering from say cardiovascular disease or diabetes because this cell type has no involvement in the development or progression of those particular diseases. If they looked at cell types related to a particular disease such as vascular endothelial cells in cardiovascular disease (Minamino et al, 2002), microglial cells in Alzheimer’s (Streit et al 2007) or pancreatic beta cells in diabetes (Sone and Kagawa, 2005) they would most likely see a decline in replicative capacity compared to age-matched controls. This was the case for lung fibroblasts in lung emphysema, demonstrated in this investigation.

Different cell types have different replicative capacities, have different functions, are maintained within different environments and thus undergo varying degrees of stresses. In addition to this, there are risk factors such as sun exposure, smoking and diet which have the potential to accelerate cellular ageing. As such, different tissues age at different rates. Therefore, the presence of disease in one tissue is not necessarily going to reflect the biological condition of another. The replicative capacity of skin fibroblasts is not necessarily going to be influenced by the presence of disease in other tissues.

A theoretical scenario where a particular disease may impact on the replicative capacity of skin fibroblasts, is if the presence of disease uses up the stem cell/progenitor cell reserve needed for cellular repair and replacement, or somehow impacts on the functioning of stem cell/progenitor cells. In this instance, damaged or lost skin cells can no longer be replaced by the stem cell/progenitor cell reserve, causing local cells to divide and replace instead. This in turn reduces the replicative capacity of those cells. This may occur in advanced stages of a disease where constant cell replacement has been undertaken. This may explain results of studies investigated in this paper which demonstrated that the replicative capacity of fibroblasts in patients with severe diabetes was diminished when compared with controls, but was insignificantly decreased in patients with mild to moderate diabetes. Also, Kuki et al (2006) has demonstrated that endothelial progenitor cells (EPCs) cultured under high glucose levels (associated with diabetes) undergo accelerated senescence. The presence of elevated oxidised low density lipoproteins (ox-LDL) observed in diabetics has also been shown to reduce the number and impair function of circulating EPCs. In addition to this, it is known that stem cells lose the capacity for self renewal when removed from the stem cell niche, suggesting that the local environment plays a crucial role in determining stem cell behaviour (Boyle et al, 2007). Therefore, the presence of diseases in advanced stages, especially those associated with inflammation, may alter the environment of stem cell niches and thus impacting on their ability to function. In this scenario, the presence of disease has the potential to impact other tissues by impairing the function of stem/progenitor cells needed for repair and maintenance.

It has often been shown that a decline in the replicative capacity of fibroblasts is correlated with an increase in chronological age of a donor. However, if the health state of donors is taken into consideration and only “healthy” subjects are investigated in this regard, there appears to be no correlation (Cristofalo et al, 1998). This suggests that the replicative capacity of a tissue only reflects biological age and not chronological age. Of course it is true, that a longer a person lives, the increased likelihood that cells become damaged, lost and replaced and this in turn would reduce the replicative capacity of those cells. However, if factors which result in cellular damage/loss such as the presence of disease (not necessarily age-related), infection or environmental factors such as smoking and sun exposure are reduced, then damage/loss of cells is reduced and the replicative capacity of those cells remains high.

Maier and Westendorp suggest an alternative explanation for the lack of relationship between donor age and replicative lifespan of skin fibroblasts: “The overall replicative capacity might decline with age but rare fibroblasts clones with extended replicative potential continue to be present at old age but do not nessesarily reflect the properties of the overall population. Therefore, the replicative capacity in vitro reflects only the expansive propagation of the longest surviving clone, which seems to have comparable in vitro characteristics when obtained from young and old individuals.”

Data on the replicative capacity of cells in regard to ageing and age-related disease is only important because the shorter the replicative capacity of a tissue, the increased likelihood that senescent cells will appear or are present. The presence of senescent cells in tissues is thought to play a role in ageing and age-related disease. Thus, it is more important to investigate the distribution and frequency of senescent cells in tissues associated with accelerated ageing syndromes, age-related diseases and chronological age.

Stuart Calimport on the subject of SENS

2009-05-26T17:23:00.012+01:00

Stuart Calimport, is a grad student at having studied Molecular Medicine at Imperial College London and Practical Ethics at The University of York . He is also a key volunteer for the Strategies for Engineered Senescence Foundation Academic Initiative.

Strategies for Engineered Negligible Senescence (SENS), by a more general definition, is an engineering strategy to reverse the effects of ageing via regenerative medicine. What it supposes is that ageing is bad (I use this term to describe negative things that reduce the complexity of complex systems) and so reversing ageing is desirable, reversing ageing is possible, and that we know just enough about ageing to cure enough of it to live a bit longer until we cure the next ageing related problem (Longevity Escape Velocity). The opposite of this viewpoint would be that ageing is a good thing, or that it is impossible to cure. This opposite view is very rarely held by gerontologists and other scientists, instead many say that ageing is bad and it is possible to cure, just not in the timespan suggested by many SENS proponents.

I find that SENS foundation (SENSF) is the only research body that I would consider both mature and progressive enough in the field, of engineering a solution to the problem of death, that I am forced to stand by it because I see no alternative except those who seem morbidly obsessed with the intricacies of death rather than those of life. Creating a separate institution somewhere in-between SENSF and the American Aging Association might be more funding friendly and attractive to more genuine scientists whilst still maintaining a life extension agenda, but too many institutions would be bad for solidarity.

I have to say that one reason that many keep coming back to is Aubrey de Grey as their reason for not thinking SENS is reasonable. Now, I am sure it is easier to put Aubrey de Grey as the leader of such a wave when invariably he attracts all sorts of characters to him who not only respect or admire him, but are looking at him to save them, in the same sort of way that many scientific intellectuals put their faith in the technological singularity. This view of Aubrey as a godhead should be propagated less by both sides as there are many scientists involved in SENS, some of which I have read plenty of high impact factor journal papers by, and no, I am not talking about Rejuvenation Research papers either. Aubrey de Grey I have seen is trying to counteract problems concerning his status at present by splitting the management of the SENSF to a number of people and referring newcomers to others instead of them asking him for answers and treating him as some sort of oracle. I am sure he also discourages linking himself to the wackier, crank characters that flock to him, instead trying to maintain more scientific networks; in just the same way that many gerontologists try to distance themselves from him. I must make it said that I feel awful writing about Aubrey as I think he has done a lot more than many scientists to capture the essence of what it is to be a scientist-to be at once intellectually responsible and an intellectual rebel.

Multiple distressing rumours concerning what without sarcasm or irony can be said to be dark maniacal schemes fit only for necromantic wizards that Aubrey and his scientific collaborators are involved in (and I am not talking about beard growing competitions) have been alluded. Can we blame the scientists spreading such rumours and hostilities? Conservative ageing scientists are scared that their already dwindling budget will be slashed further, but creating rumours about the things politicians and the public most fear will surely do no good to themselves. Concerning such rumour mongering I am sure the rest of the gerontological community would be happy to help slay the dragon they have created out of Aubrey by laying waste to any unfounded rumours at once.

As a final point I would like to list some pros of SENS to balance all the cons of such a risky endeavour: It encourages more theoretical biology, biotech innovation and interdisciplinary study. I find too much of molecular biology to be scripted and for nobody to really question how problems are being tackled. Other areas such as cancer research could learn a lot from the thought processes behind SENS, even if they are loath to think about WILT, which I do not blame them for.

Mindless screens of genes and proteins do not provoke or inspire innovation. It is only when someone says that, fundamentally, that they do not like what is going on, so lets go back to the drawing board and rethink the whole scenario that paradigm shifts (an overly misused phrase) occur.

Microarray analysis of senescent keratocytes (EK1.Br)

2009-05-20T20:20:00.008+01:00

The study below carried out microarray analysis of senescent human fibroblastoid keratocytes (EK1.Br), which demonstrated that, in contrast with other fibroblast strains, senescence in this instance does not appear to be associated with a shift to a catabolic phenotype. The great thing about this paper is that it has provided a link to a fully searchable public access database, so you can explore the data yourself (www.madras.cf.ac.uk/cornea).

A transcriptomic analysis of the EK1.Br strain of human fibroblastoid keratocytes: The effects of growth, quiescence and senescence

David Kipling, Dawn L. Jones, S. Kaye Smith, Peter J. Giles, Katrin Jennert-Burston, Badr Ibrahim, Angela N.P. Sheerin, Amy J.C. Evans, William Rhys-Willams and Richard G.A. Faragher,

Abstract

There is a growing need within ocular research for well-defined cellular models of normal corneal biology. To meet this need we created and partially characterised a standard strain of human fibroblastoid keratocytes (EK1.Br) and demonstrated that phenotypic changes occur within these cells with replicative senescence in vitro. Using Affymetrix HG-U133A oligonucleotide arrays, this paper reports both a comprehensive analysis of the transcriptome of EK1.Br in the growing, quiescent and senescent states and a comparison of that transcriptome with those of primary corneal endothelium, lung fibroblasts and dermal fibroblasts grown under identical conditions. Data mining shows (i) that EK1.Br retain the characteristic transcriptional fingerprint of keratocytes in vitro (ii) that this phenotype can be distinguished from those of other ‘fibroblasts’ by groups of highly differentially expressed genes and (iii) that senescence induces a distinct dedifferentiation phenomenon in EK1.Br. These findings are contextualised into the broader literature on replicative senescence and are supported with a web-accessible and fully searchable public-access database.

www.madras.cf.ac.uk/cornea

Cellular senescence in pharmacogerontology research

2009-05-16T17:39:00.005+01:00

The administration of pharmacological agents to older persons often results in a higher incidence of drug toxicity and adverse drug reactions compared with the young. This is mainly due to changes in pharmacokinetic (the process by which a drug is absorbed distributed, metabolised, and eliminated by the body) and pharmacodynamic (what a drug does to the body) properties believed to be the result of biological alterations linked to the ageing process. Therefore, understanding the mechanisms of ageing, the biological alterations they bring about and the biological consequence of such alterations could help answer questions concerning the pharmacokinetic and pharmacodynamic changes observed in the elderly.

The pharmacokinetic changes observed in elderly patients are well understood (click here) and allowances can be made for them. However, pharmacodynamic’s is much harder to predict as it requires an understanding of the biological changes associated with ageing (many of which may be individual specific). Insight into these processes has mainly been generated by laboratories focused on the molecular mechanism underlying the ageing process. These groups often have a limited understanding of the pharmacology of the elderly. Therefore, research in this area appears not to have progressed beyond cataloguing the observed drug responses in the elderly.

The accumulation of senescent cells in tissues has been linked to ageing and disease and as such could potentially alter the biological response to drugs in the elderly. When a cell becomes senescent, it undergoes a radically altered phenotype (click here). Microarray analysis of primary human lung fibroblasts (IMR-90) and primary skin fibroblasts (Detroit 551) reported that 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 in the transcriptome is akin to that seen when cells are induced to differentiate (Truckenmiller et al 2001). Essentially, senescent cells should be treated as a completely different cell type from when they were growth competent. Therefore, more research should be carried to determine whether or not senescent cells display an altered responsiveness to pharmacological agents.

Conclusion

By bridging the gap between pharmacokinetic and pharmacodynamic studies and molecular gerontology it is hoped that pharmaceutical intervention might one day be more precisely targeted to the age of the patient (and thus, the biological status of the target tissue). It is anticipated that the development of in-vivo and in-vitro models of tissue ageing will facilitate the necessary advances in pharmacogerontology.

See: Bridging the gap: ageing, pharmacokinetics and pharmacodynamics

Cellular senescence papers: different cell types

2009-05-11T17:37:00.016+01:00

The following is a list of papers demonstrating cellular senescence in cell types other than fibroblasts. It will gradually be up-dated.

Endothelial cells

Vascular endothelial senescence: from mechanisms to pathophysiology. Erusalimsky JD. J Appl Physiol. 2009 Jan;106(1):326-32. Epub 2008 Nov 26.

Telomere attrition and accumulation of senescent cells in cultured human endothelial cells. Hastings R, Qureshi M, Verma R, Lacy PS, Williams B. Cell Prolif. 2004 Aug;37(4):317-24

Endothelial Cell Senescence in Human Atherosclerosis. Minamino et al. Circulation. 2002;105:1541.)

A cell kinetic analysis of human umbilical vein endothelial cells. Kalashnik et al. Mech Ageing Dev. 2000 Dec 1;120(1-3):23-32.

Vascular smooth muscle cells

Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress. Matthews et al, Circ Res. 2006 Jul 21;99(2):156-64. Epub 2006 Jun 22

Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification. Burton et al (2009) Experimental gerontology 2009 Oct;44(10):659-65 PubMed ID:(19631729)

Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition. Nakano-Kurimoto et al, Am J Physiol Heart Circ Physiol. 2009 Sep 11. [Epub ahead of print]

Epithelial cells

Beta-galactosidase histochemistry and telomere loss in senescent retinal pigment epithelial cells. Matsunaga et al, Invest Ophthalmol Vis Sci. 1999 Jan;40(1):197-202

T-cells

T cell replicative senescence: pleiotropic effects on human aging. Effros RB, Ann N Y Acad Sci. 2004 Jun;1019:123-6

The role of CD8+ T-cell replicative senescence in human aging. Effros RB, Dagarag M, Spaulding C, Man J. Immunol Rev. 2005 Jun;205:147-57

Microglia

Microglial senescence: does the brain's immune system have an expiration date? Streit WJ Trends Neurosci. 2006 Sep;29(9):506-10. Epub 2006 Jul 20

The role of microglial cellular senescence in the aging and Alzheimer diseased brain. Flanary B, Rejuvenation Res. 2005 Summer;8(2):82-5

Astrocytes

Astrocytes aged in vitro show a decreased neuroprotective capacity. Pertusa et al, J Neurochem. 2007 May;101(3):794-805. Epub 2007 Jan 23

Osteoblasts

Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts. Kassem et al. Osteoporos Int. 1997;7(6):514-24.

Relationship between periarticular osteoporosis and osteoblast senescence in patients with rheumatoid arthritis. Yudoh K, Matsuno H, Kimura T., Clin Calcium. 2001 May;11(5):612-8

Chondrocytes

Aging, articular cartilage chondrocyte senescence and osteoarthritis. Martin and Buckwalter, Biogerontology. 2002;3(5):257-64

Pancreatic Beta cells

Pancreatic beta cell senescence contributes to the pathogenesis of type 2 diabetes in high-fat diet-induced diabetic mice. Sone H, Kagawa Y., Diabetologia. 2005 Jan;48(1):58-67. Epub 2004 Dec 29

Hepatocytes

Role of replicative senescence in the progression of fibrosis in hepatitis C virus (HCV) recurrence after liver transplantation. Trak-Smayra et al, Transplantation. 2004 Jun 15;77(11):1755-60

Hepatocyte telomere shortening and senescence are general markers of human liver cirrhosis. Wiemann et al, FASEB J. 2002 Jul;16(9):935-42

Renal cells

Increased expression of senescence-associated cell cycle inhibitor p16INK4a in deteriorating renal transplants and diseased native kidney. Melk et al, Am J Transplant. 2005 Jun;5(6):1375-82

Stem/Progenitor cells

Replicative senescence of mesenchymal stem cells: a continuous and organized process. Wagner et al, PLoS ONE. 2008 May 21;3(5):e2213

Premature senescence of highly proliferative endothelial progenitor cells is induced by tumor necrosis factor-alpha via the p38 mitogen-activated protein kinase pathway. Zhang et al, FASEB J. 2009 May;23(5):1358-65. Epub 2009 Jan 5

DISEASE FOCUS: Alzheimer’s

2009-05-05T21:02:00.006+01:00

Introduction

Alzheimer’s disease (AD) is the most common form of dementia and is found predominantly in people aged over 65 years. It is progressive, degenerative and currently irreversible. Features of the disease include memory loss, decreased reasoning and judgment and changes in mood, behaviour and personality. The majority of the research into AD is focused on protein plaques (mostly made up of a protein called B-amyloid) and neurofibrillary tangles (composed of a protein called tau) found in the brain of AD patients. The role of amyloid plaques and neurofibrillary tangles on the functioning of the brain is poorly understood and research is ongoing. This article however, neither focuses on plaques or tangles, but instead on the role of cellular senescence (specifically microglial and astrocytes) in the development and/or progression of AD.

Microglial cells and Astrocytes in Alzheimer’s

Both microglial and astrocytes are mitotic cells which have been shown to undergo cellular senescence (Streit, 2006 and Pertusa et al, 2007). Moving around the brain, microglia function as immune cells to remove damaged neurons, plaques and infecting micro-organisms (Pivneva, 2008). Astrocytes appear to have numerous responsibilities including providing nutrients to neurons and neuronal maintenance (Seth and Koul, 2008, Rodriguez et al 2009). Therefore, without microglia and astrocytes, or a decline in their function, there would most likely be an increase in neuronal damage and this could manifest as disease. An accumulation of senescent microglial and astrocytes in the brain could lead to such a functional decline. However, as with a number of cell types, little is known about the senescent-specific phenotype of microglia and astrocytes and little work has been carried out to investigate the possible contribution senescent cells may have on the development/progression of AD.

An interesting study by Streit et al (2007) demonstrated that the presence of amyloid protein promotes cellular senescence in microglial cells. Amyloid protein causes microglial cells to become activated and thus proliferate to facilitate amyloid removal. Constant activation and cell turnover of microglial cells would result in gradual telomere attrition and thus an increased appearance of senescent cells. Apolipoprotein E (apoE) also plays a role in the degradation and clearance of amyloid protein by astrocytes. However, varients of this gene have been shown to be a major risk factor in the development of late onset AD (Wang and Ding, 2008). Research has shown that this varient enhances the production of amyloid protein (Ye et al, 2005), which may consequently lead to an increase in microglial activation and accelerated appearance of senescent microglia. The presence of amyloid is just one example of how microglial cell senescence may become accelerated, but other currently unknown risk factors may also have the same impact.

Little is known about the mechanisms by which astrocytes become activated and it can only be speculated as to the effects senescent astrocytes would have on the brain (if any). Astrocytes have been shown to become activated and proliferate in culture in the presence of cytokines and growth factors (Selmaj et al 1990) and by neuroinflammation in the brain (Norris et al, 2005). General features of a senescent phenotype appear to be an up-regulation of pro-inflammatory cytokines, growth factors and matrix degrading proteins. If this is true for senescent microglial cells, then it could be speculated that the accumulation of senescent microglial cells (if they persist in tissue) may consequently lead to the activation and proliferation of astrocytes. Interestingly, interleukin 1 (a cytokine known to stimulate astrocyte proliferation) is elevated in both Down syndrome (risk factor for AD) and in AD (Griffin et al, 1989). This up-regulation of interleukin 1 appears to orinate from activated microglial (Mrak, 2001). Inflammaotory mediators have long been shown to be up-regulated in pathologically vulnerable regions of the brain in AD (Rogers, 2008). The constant proliferation of astrocytes would eventially result in the accumulated appearance of senescent astrocytes (further research needs to be carried out to determine if astrocyte numbers increase in AD progression). This means that neurons will become functionally impaired, damaged or lossed. Pertusa et al (2007) found that aged astrocytes in vitro show a decreased neuroprotective cacpacity. Long-term cultures of astrocytes demonstrated positive staining for senescence-associated-beta-galactosidase (a senescent marker, Dimri et al, 1995) suggesting that this functional decline is associated with the senescent phenotype of astrocytes.

Neuronal loss may not be a major problem initally since they are most likey replaced by neural stem/progenitor cells (Taupin, 2006). However, these cells have also been shown to undergo cellular senescence or become functionally impaired with age (Sharpless and DePinho 2007, Ruzankina and Brown 2007). This means, the brain would reach a point where neurons are being lost without replacement.

A theoretical consequence of the senescent astrocyte phenotype might be related to that which is observed in senescent vascular endothelial cells (ECs). Senescent vascular ECs show a reduction in nitric oxide (NO) production by eNOS (Minamino et al, 2002). In the vascular system, NO signals the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow. A reduction in NO would therefore restrict bloodflow and reduce oxygen supply to much needed tissues. NO reduction has been suggested to be a significant risk factor for cardiovascular disease.

Astrocytes perform many functions, including biochemical support of endothelial cells which form the blood-brain barrier. eNOS activity has also been shown to be present in astrocytes (Lin et al, 2007), but the impact of cellular senescence (if any) on eNOS activity on this cell type is currently lacking. However, since it has been shown that astrocytes play a direct role in controlling blood flow in the brain (Koehler et al 2009) and if senescent astrocytes do reduce the synthesis of NO, then this may lead to a reduction of blood flow to certain areas of the brain. This means less oxygen and potential cell death..

One final point of mention. In AD, the hippocampus (important in long-term memory) and the neocortex (higher level cognitive function such as language, learning and memory) are one of the first areas of the brain to undergo damage (Scheff and Price, 2006). Interestingly, both microglial and astrocytes have been shown to specifically proliferate in the hippocampus and temporal neocortex following global cerebral ischemia in young adult monkey brain (Tonchev et al, 2003). This region specific proliferation of microglial cells and astrocytes, the same region affected in AD, provides some link that these two cell types may play a role in the pathogenesis of AD.

Conclusion

To date little work has been carried out to investigate microglial cell and astrocyte senescence in AD. Little is known about the senescent phenotype of microglial cells and astrocytes and what impact (if any) this phenotype may consequently have on the brain. A number of points in this article can only be speculative, but based on what we know about the phenotype of other senescent cell types and the theoretical impact of their presence, it is not difficult to envisage a role for cellular senescence in AD development and/or progression.

ALSO SEE:

Phenotypic changes associated with cellular senescence

DISEASE FOCUS: Atherosclerosis and vascular calcification
.

Cellular senescence in disease states

Cellular senescence, ageing and disease (2009)

Chris Patil: Ageing Research and the Media

2008-06-19T18:19:00.007+01:00

Chris Patil of Ouroboros is a postdoctoral fellow, currently working with Judith Campisi in the Life Sciences division of the Lawrence Berkeley National Lab. Here is Chris's response to the question I recently sent to a number of biogerontologists:

The main purpose of ageing research at present is NOT to make people young and immortal as is often publicised in the media, but instead to prevent/combat disease and disability, allowing everyone to live healthier lives for longer. Is this media representation of ageing research detrimental to the true focus of ageing research? If you disagree with the main purpose of ageing research outlined in the question please state why?

I don’t believe in a monolithic entity called “aging research”, and consequently I don’t believe that that entity has a “true focus”. Aging is a huge field; there are any number of ways to engage with it; there is consequently tremendous diversity among scientists who consider themselves to be involved in aging research. There are distinct sub-communities, certainly, which can be classified according to their priorities and focus; in comparisons between these sub-communities, patterns do emerge. For instance, I’ve noticed a tradeoff between immediacy and scope — that is, those whose work can benefit elderly people today tend to have fairly modest ambitions compared to those whose labors will take some time to bear fruit. A lot of this has to do with individual priorities, and where individual scientists feel like they can do the most good and/or do the work that makes them happiest.

I feel most comfortable speaking for myself and my community — let’s call us “academic biogerontologists” -- whom I’m going to (conveniently) define as the set of individuals devoted primarily to the production of fundamental research in the biology of aging, who primarily work in universities or similarly organized institutions, and whose work is primarily published in peer-reviewed journals.

For academic biogerontologists, there are two related aims of aging research. The explicit, near-term goal is improving our understanding of the aging process at multiple levels — at the cellular and molecular levels, but also (as tools get better) at the level of cell-cells interactions (i.e., tissues) as well as the gemisch of global gene expression studies that fall under the “systems biology” umbrella. The (occasionally) implicit, longer-term goal is to use this understanding to create interventions that will improve the health and happiness of human beings — and here the ambitions range from the treatment of single aging-related diseases to therapies that will delay or even reverse the aging process itself.

For us, the two processes go hand in hand, and both are essential. Fundamental research into the mechanisms of aging is constantly revealing new connections between well-studied systems (like nutritional control of cell growth) and the basic biology of aging. It is from these connections, from this knowledge, that the interventionist tools of the future will emerge. Both sorts of work can and should be undertaken simultaneously, if not by the same groups then in an open community where there is a great deal of communication across the aisle, so that both types of scholars are learning from each other at an optimum rate. Indeed, we’re beginning to see some of the first pharmaceuticals developed by academic-industrial collaborations and academic spinoff companies; it’s an exciting time.

One thing that we are constantly learning from basic studies is that as much as we know (especially, as much as we’ve learned in the past 15 years or so, a period that I think of as the dawning era of the modern biology of aging), we could always know more. One example is the recent discovery in model organisms that stem cells don’t function well in aged microenvironments — in aged niches, they either don’t regenerate well or become dysregulated; for those of you just joining us, having dysregulated telomerase-positive immortal cells in your body is potentially a fairly bad thing. These recent observations have caused us to seriously rethink the strategies that will be required to effectively use stem cell transplantation in the treatment of age-related disease. So I don’t think we’re ever going to reach the point where we can take off our gloves and say, “This is a solved problem; no more fundamental research for us!” Not that I think anyone is seriously advocating a moratorium on future basic studies, any more than they were advocating injecting old people with huge doses of undifferentiated cells. There are differences of opinion on the relative import of basic vs applied work but inasmuch as both fields are far smaller than they should be I don’t think the time has arrived for debating tradeoffs between the two.

But I’ve digressed extensively. Do I think that media representations are positive or negative? To the extent that they spread the word in a measured way, like the piece in the Economist earlier this year. I think they’re largely positive. Even when individual articles get their “zing” from focusing on what I consider to be quite long-term goals, I think they still do a tremendous amount of good by raising consciousness about the biology of aging. We’re entering a period of history when people will become more and more willing to appreciate the benefit of long-term thinking, especially as related to technology — we’re already seeing that with the environment, and I think aging research and anti-aging medicine will be another example. One of my scientific heroes, Carl Sagan, popularized space exploration and even the nascent field of exobiology by making bold statements about the longest-term and wildest possibilities, and in so doing he inspired a generation of young scientists. I don’t see any reason why aging should be different: As long as we’re scientifically responsible, honest about the current state of affairs, and reasonable in our predictions, why shouldn’t we emphasize the long-term payoff of our work? Full steam ahead!

Chris

Ouroboros

Anti-Ageing Research Blog

2008-06-16T23:21:00.003+01:00

Soon to be up and running is my new blog, anti-ageing research, which focuses NOT on the biology of ageing, but the ideas and research aimed at preventing or treating the effects of the ageing process. Check it out.

Cheers

Dom

http://anti-ageing-research.blogspot.com/

Aubrey de Grey: Ageing research and the media

2008-06-12T00:54:00.011+01:00

I recently emailed a number of biogerontologists the following question:

One of the first people to respond to this question was Aubrey de Grey with the following:

Your premise is somewhat wide of the mark, in that most people who do research into aging actually regard any appreciable therapeutic benefit from their work as a very remote possibility (in both senses - unlikely, and very distant in time if it happens at all). Thus, their purpose is merely to **understand** aging, rather in the way that the purpose of meteorologists is to understand the weather, as opposed to actually doing anything about it.

However, there is indeed a small but growing minority of gerontologists (including myself) who do identify the postponement of aging as our main purpose, not least because we are more optimistic than the majority of our colleagues with regard to the possibility of success. For us, you have it exactly right: the goal is to prevent people from going downhill as they become chronologically older. The problem is, we recognise that this will have a side-effect (which most of us regard as a side-benefit, but that's another issue altogether): people won't tend to die peacefully in their sleep either, any more than healthy 30-year-olds do, until and unless they get to an age at which the therapies we develop cease to work. Worse yet (he said, sarcastically), for those of us (like myself) who claim that the therapies that will first make a major impact on aging will be bona fide rejuvenation therapies, i.e. therapies that restore the molecular and cellular (and higher-order) structure of the body to something like the way it was in young adulthood, the situation is particularly extreme, because the likely rate at which such technologies will be improved following their initial development is such that the therapies will never cease to work: aging will be postponed faster than it occurs, so it will never catch up with us. (This is the phenomenon that I've termed "longevity escape velocity".) Thus, I predict that people's life expectancies will be determined only by their incidence of death from causes not related to their age, like accidents and nearby supernovae. That is the simple and inescapeable conclusion of the work I do. Clearly it means people still have a non-zero risk of death each year (or indeed each day) - but unfortunately it does sound awfully like immortality if you're the sort of journalist who wants to sell papers, so that's how it tends to get described.

So to your main question: is this detrimental? Yes, I believe it is immensely detrimental. (I don't precisely blame the journalists in question, you understand - they're just doing what they're paid to do - but still.) Ultimately, the reason why calling my goal "immortality" sells papers is because it trivialises it - it confuses my work with something that we all know is impossible, i.e. the technological elimination of any risk of death. And an awful lot of people need that confusion - they need to be helped to believe that what I'm doing is really not science but just entertainment. Why do they need that? Because they've made their peace with aging. They've spent their lives in the situation where there was no hope for escaping this terrible, yet rather distant, fate - so they've had the choice of either (a) spending their time preoccupied by that, or (b) putting it out of their minds and getting on with their miserably short lives, making the best of a bad job. And of course the rational thing to do in such a situation, even if it entails quite unbelievably irrational rationalisations, is (b). So now this troublemaker comes along and says there may be a chance. Now, if I were saying "Hey, here is the actual therapy, today, proven and provided", there'd be no problem - just as when Pasteur worked out that hygiene was a good idea, or whatever. But unfortunately all I'm offering is a **chance** that in a few **decades** we will have that techniology. And an awful lot of people don't want to get their hopes up, for fear of having them dashed.... so they stick to what they know, their faith that aging really is still inevitable. But at the same time, they desperately want, in their heart of hearts, to know as soon as possible when breakthroughs are made - which is why I do at least two media interviews every WEEK even though I don't even do any experiments of my own. But that exposure to my work needs to be camouflaged as entertainment in order to be palatable.

So, clearly, what I would dearly like to occur is for the media to have the guts to tell my story like it is, and to dare/embarrass/coerce their audience into thinking about it properly and then getting off their backsides and contributing whatever they can (money, activism, whatever) to the crusade to save 100,000 lives per day. But as I said, the journalists in question don't get paid to make their audience uncomfortable, so I'm not holding my breath.

Cheers,

Aubrey

http://www.mfoundation.org/aging2008/

Immune response to cancer cells (and maybe senescent cells)

2008-06-09T17:51:00.005+01:00

Since any immune response to the presence of senescent cells may possibly be similar to that of cancer cells, the following is a brief outline describing the key points in the removal process (Ullrich et al, 2007, Vulink et al, 2008, Wesa and Storkus, 2008, Chan and Housseau, 2008).

Dendritic cells are antigen-presenting cells found in all tissues of the body and are crucial for stimulating a naïve T-lymphocyte response in the removal of tumour cells.

In the presence of tumour cells, dendritic cells capture (by engulfing portions of the tumour cell) and process tumour-specific molecules (antigens) so that they become presented on their cell surface. Dendritic cells start to mature as they migrate to the lymph nodes, a process which enables dendritic cells to present the tumour information. The maturation process is needed as additional co-stimulatory molecules are required so that they can be recognised by other immune cells. When mature dendritic cells reach the lymph nodes, they interact with cytotoxic T-lymphocytes (CTLs), and pass on the tumour information, causing CTLs to become activated and consequently proliferate. The large numbers of CTLs then circulate the body, recognising the tumour-specific antigens, binding to them and destroying tumour cells by the release of enzymes.

If a cancer cell (or a senescent cell) is not removed by the immune system, then something isn’t working as it should. From the brief overview above, there are a number of points between cell recognition and removal that could have failed. These are:

(1) Dendritic cells did not recognise the cancer/senescent cell.
(2) The dendritic cells did not display cancer/senescent specific markers on it’s surface.
(3) Lymphocytes were not activated in response to dendritic cells.

The changes which occur as we age which may have an impact on the removal of tumour cells/senescent cells will be discussed next.

www.springerlink.com/index/JW847G541L435103.pdf

Why do senescent cells accumulate in tissues?

2008-05-28T00:53:00.007+01:00

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?

2008-05-22T00:24:00.005+01:00

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?

2008-05-16T18:17:00.009+01:00

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 result 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

2008-05-16T13:46:00.003+01:00

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

2008-05-16T00:59:00.012+01:00

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

2008-05-12T17:25:00.020+01:00

Cellular Senescence and vascular calcification

.
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).

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.

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.

SEE: Burton et al (2009) Microarray analysis of senescent vascular smooth muscle cells: A link to atherosclerosis and vascular calcification

DISEASE FOCUS: Atherosclerosis and vascular calcification

2008-05-09T01:37:00.004+01:00

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

2008-05-07T13:35:00.004+01:00

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.

Cellular senescence and atherosclerosis

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

2008-05-05T14:33:00.006+01:00

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

2008-05-02T21:57:00.004+01:00

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.

Cellular senescence in disease states

2008-04-29T01:30:00.005+01:00

In some instances, cellular senescence is thought to contribute to the development and/or progression of age-related disease, but in others, the presence of disease may accelerate the accumulation of senescent cells.

A recent study has provided strong evidence to suggest that intervertebral disc degeneration, a major cause of low back pain, is due to accelerated cellular senescence (Le Maitre et al, 2007). Cells isolated from normal and degenerate human tissue were assessed for mean telomere length, SA-β-Gal, and replicative potential. Mean telomere length decreased with age in cells from non-degenerate tissue and also decreased with progressive stages of degeneration. SA-β-Gal staining was not observed in non-degenerative patients unlike cells from degenerative discs which did exhibit 10-12% SA-β-Gal staining and decrease in replicative potential. However, the factors which may have led to accelerated senescence in this instance was not discussed. There are three possible reasons why cellular senescence was accelerated in this instance; (1) Unknown factors resulted in the damage and removal of cells, resulting in cell turnover for replacement, (2) ROS were involved causing stress induced premature senescence (SIPS) or (3) telomeres in these cells for some unknown reason started off shorter than normal, meaning less cell turnover is required for the appearance of senescent cells.

Other studies have shown a correlation between disease states and the presence of senescent cells in vivo. SA-β-Gal staining was used to detect senescent cells in normal liver, liver with chronic hepatitis C and hepatocellular carcinoma (HCC) (Paradis et al, 2001). They found senescent cells present in 3 of 15 (20%) normal livers tested, 16 of 32 (50%) in livers with chronic hepatitis C and in 6 of 10 (60%) livers with HCC. The presence of senescent cells in normal livers was found to be associated with old age. Interestingly, the presence of senescent cells in non-tumoural tissues was strongly correlated with the presence of HCC in the surrounding liver. This demonstrates not only that the ageing of one tissue can have a direct impact on another but also as suggested by Judith Campisi, senescent cells may contribute to carcinogenesis (Campisi, 1997).

Another study looked at cellular senescence in human benign prostatic hyperplasia (BPH) specimens (Choi et al, 2000). BPH is a disease associated with an abnormal growth of the adult prostate that begins mid to late life. Results from this study found that 40% of the analysed samples showed positive staining for SA-β-Gal and only in the epithelial cells. A high prostate weight (> 55g) was found to correlate strongly with the expression of SA-β-Gal. Prostates weighing less than 55g tended to lack senescent epithelial cells. It was suggested that the accumulation of senescent epithelial cells may play a role in the development of prostatic enlargement associated with BPH. However, the accumulation of senescent cells in this case is likely to be a consequence of the disease, which may lead further to its progression. The enlargement of the prostate may be the result of unregulated stimulated proliferation, increasing cell turnover and consequently the appearance of senescent cells. This may explain why a stronger expression of SA-β-Gal is detected in prostates weighing more than 55g since they may have undergone more cellular divisions.

During the pathogenesis of type 2 diabetes, insulin resistance causes compensatory proliferation of pancreatic beta cells. This compensatory proliferation might accelerate cellular senescence contributing further to the progression of diabetes. To investigate this, one group used nutrient-induced diabetic mice to analyse beta cells for SA-β-Gal and the proliferation marker Ki67 (Sone and Kagawa, 2005). At 4 months, the proliferation of beta cells was 2.2 fold higher than in the control group. At 12 months, the frequency of Ki67 decreased to one-third that of the control and SA-β-Gal positive cells increased to 4.7 fold that of the control group. This increase in the senescent beta cell fraction correlated with insufficient insulin release, suggesting cellular senescence may contribute to diet-induced diabetes. In this instance it is difficult to determine whether senescence is the cause or the consequence of insulin resistance. It later appears to be a contributor but whether it is also the initiating factor is unknown.

An increase in the number of senescent primary lung fibroblasts has also been shown to increase in patients with emphysema compared with normal controls (Muller et al, 2006). An average of 4% of cells from control patients stained positive for SA-β-Gal compared to an average of 16% in patients with emphysema. It is possible that long-term exposure to tobacco smoke accelerates the formation of senescent cells, which subsequently may lead to loss of elasticity of the lung tissue, destruction of structures supporting the alveoli, and destruction of capillaries feeding the alveoli observed with emphysema. One study has shown for example that cigarette smoke induces senescence in alveolar epithelial cells (Tsuji et al, 2004).

Another study used senescent associated p16 instead of SA-β-Gal to detect senescent cells in kidneys with glomerular disease (GD) (Sis et al, 2007). Glomerular diseases include many conditions which fall into two major categories; Glomerulonephritis describes the inflammation of the membrane tissue in the kidney that serves as a filter, separating wastes and extra fluid from the blood. Glomerulosclerosis is the scarring or hardening of the tiny blood vessels within the kidney. This study found an increased expression of the nuclear p16 in samples with GD compared with normal. Independently, older age and interstitial inflammation was associated with increased expression of nuclear p16. Since senescent cells adopt a pro-inflammatory phenotype, their presence may be a contributing factor in inflammation observed in glomerulonephritis.

All these examples demonstrate the presence of senescent cells not only in vivo, but more specifically in disease states. This suggests that the accumulation of senescent cells in normal tissues is the result of injuries to those tissues or in some cases unregulated stimulation of proliferation. This suggests that if no injuries occurred as a consequence of disease, environmental factors or by normal biological/mechanical wear and tear, ageing of mitotic tissues would be greatly reduced.

Whether cellular senescence is the cause or the consequence of some diseases is yet to be answered.

www.springerlink.com/index/JW847G541L435103.pdf

Replicative Senescence in vivo

2008-04-29T01:25:00.001+01:00

The early observation that young cultured fibroblasts have a higher growth potential than those derived from adults led to the proposal that senescent cells may play a causal role in organismal ageing (Hayflick, 1961). For this to be the case, senescent cells need not only to be shown to be present in living tissue but also to persist for long periods. However, cellular senescence at the time of Hayflick’s proposal was thought by many to be a tissue culture artefact, with no relevance to normal human ageing. Since the growth conditions of cultured cells are dissimilar to that found in vivo, it was thought that these differences resulted in the formation of senescent cells in culture but not in vivo. It has also been argued that if senescent cells are present within tissues, the fraction would be so small that they are unlikely to have any impact on the surrounding tissue and ageing in general.

At the time of Hayflicks proposal there was no way of detecting senescent cells in vivo. It wasn’t until over 30 years later that a marker was used to demonstrate the presence of senescent cells in human dermis in vivo (Dimri et al, 1995). This detection system is based on a modified beta-galactosidase assay and is termed senescence-associated beta-galactosidase (SA-β-Gal). Skin samples were taken from 20 human donors aged 20-90 years, sectioned and stained for SA-β-Gal. Results showed an age-dependent increase in SA-β-Gal staining in dermal fibroblasts and epidermal keratinocytes. None of the young donors (<40yr)>69 yr) donor did display positive staining. About half of the young donors showed some epidermal staining whereas positive staining was always observed in the epidermis of old donors. Using the same technique to identify senescence cells, another group looked at senescence of the retinal pigment epithelium (RPE) of Rhesus monkeys (Hjelmeland et al, 1999). Results also showed an accumulation of SA-β-Gal positive cells in the eyes of older monkeys. Another study also found little or no SA-β-Gal staining in HCECs of corneas from young donors but was easily detected in corneas from older donors (Mimura and Joyce, 2006).
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More recent studies have used other markers to detect cellular senescence in mitotic tissues (Herbig et al, 2006, Jeyapalan et al, 2007). These studies investigated cellular senescence in the tissues of ageing primates. They used markers of senescence such as telomere damage, active checkpoint kinase ATM, high levels of heterochromatin proteins and elevated levels of p16 in skin biopsies from baboons with advancing age. The number of dermal fibroblasts containing damaged telomeres reached a value of over 15% of total fibroblasts in very old animals (26-30 years) compared to young (5-6 years) where DNA damage were rarely detected. However, in skeletal muscle, a postmitotic tissue, only a small percentage of damage to telomeres was detected regardless of age.

Replicative senescence at the molecular level in vitro

2008-04-24T14:41:00.004+01:00

Cell cycle independent senescence (CCIS)

In contrast to the TDS and TIS mechanisms, cell cycle independent senescence (CCIS) does not seem to require cell division. A number of signals have been shown to trigger CCIS, including oxidative stress (Von Zglinicki et al, 2000), histone deacetylase inhibitors (Munro et al, 2004), and expression of some activated oncogenes such as ras, raf and MEK (Di Micco et al, 2006). This section focuses on oxidative stress and activated oncogenes.

The proposal that oxidative stress may cause CCIS comes from studies that have treated cells with concentrations of H2O2 and found that those cells enter a long-term growth arrest resembling replicative senescence; often called stress induced premature senescence or SIPS. One such study treated human fibroblasts with sub-lethal concentrations of hydrogen peroxide which induced cell cycle arrest, with an initial 2-3 fold increase in the level of p53 protein and subsequently an increase in the level of p21 protein (Chen et al, 1998). One study has shown that the up-regulation of Caveolin-1 by oxidative stress is required to induce premature senescence (Volonte et al, 2002). Caveolin-1 is thought to function as a “transformation suppressor” protein. For example Caveolin-1 mRNA and protein expression are lost or reduced during cell transformations by activated oncogenes.

Oxidative stress is thought to result in damage to DNA and DNA mutations. The most severe type of DNA damage is double-strand breaks (DSBs). Studies have shown that cells treated with agents that cause DSBs lead to an increase in p16 expression and premature senescence (Robles and Adami, 1998). Induction of DNA damage resulted in the induction of p53 and p21. The concentration of p21 protein increases 100-fold and then begins to drop, followed by an increase in p16. This is similar to what occurs during replicative senescence.

Oxidative stress is also thought to result in DNA becoming mutated and this may impact oncogenes such as RAS. Studies have shown that mutated oncogenic Ras can result in a permanent G1 arrest (Serrano et al, 1997). Oncogenic Ras is commonly associated with the transformation of primary cells to an immortal state. It seems, however, that this transformation can only occur with either the co-operation of another oncogene or the inactivation of tumour suppressers such as p53 or p16. Serrano and co-workers also found that Ras induced cell senescence is accompanied by the accumulation of p53 and p16 and that the inactivation of either one of these prevents Ras induced arrest in rodent cells. More recent studies have also shown that Ras can induce cell senescence in vascular smooth muscle cells (Minamino et al, 2003) and endothelial cells (Spyridopoulos et al, 2002). In the first study, an activated ras allele was introduced into human vascular smooth muscle cells using retroviral infection. This resulted in growth arrest with phenotypic characteristics of cell senescence. In the second study, bovine aortic endothelial cells were infected using the adenovirus containing the activated ras gene. Over-expression of ras in this case led to G1 and G2/M-cell cycle arrest after 72 hours due to induction of p21. p21 induction again appears to be the initiating factor of Ras induced senescence and induction of p16 is required to maintain the senescent state. One group for example found that Ras was capable of causing growth arrest in both p21 and p53 negative human fibroblasts, suggesting Ras is activating the p16 pathway (Wei et al, 2001).

An overview of telomere-dependent, telomere-dependent and cell-cycle independent senescence is shown below.

Replicative senescence at the molecular level in vitro

2008-04-23T14:50:00.001+01:00

Telomere-independent senescence (TIS)

Like p21, p16 is a CDK inhibitor involved in cell cycle arrest. Over expression of p16 causes G1 arrest in early passage cells by inhibiting the phosphorylation of the retinoblastoma protein (Kato et al, 1998). However, the removal of p16 activity results in only minimal lifespan extension that was terminated by senescence (Wei et al, 2003). p16 plays a role in maintaining senescence triggered by a short telomere. It appears that p21 is the initiating factor in the growth arrest observed in senescent cells, but p16 is required to maintain that state. p21 has been shown to initially increase in fibroblast cultures but later gradually decrease (Alcorta et al, 1996). During the decline of p21, p16 protein levels gradually increased in senescent cultures, reaching nearly 40-fold higher than in early cultures.

P16 is thought to trigger senescence independently of telomere length. For example, the inactivated p53 in two different fibroblast strains: WI-38 (from foetal lung) and BJ (from neonatal foreskin) resulted in the continuation of growth without Rb inactivation in BJ cells, whereas WI-38 cells fail to grow even when both Rb and p53 are inactivated. It was suggested that these two outcomes was due to the intrinsic differences in the ability to induce p16 at senescence. This proposal was based on the understanding that like WI-38, other human cells appear to undergo replicative senescence prior to telomere shortening by the induction of p16 (Itahana et al, 2003). The ability of p16 to cause growth arrest even after Rb inactivation also suggests that p16 can function independently of Rb activity. Further support for a telomere-independent mechanism was provided from studies which have shown that telomerase activity alone was insufficient to extend the replicative potential of human keratinocytes or mammary epithelial cells (Kiyono et al, 1998). It was shown that down-regulation of p16 in combination with telomerase activity did lead to replicative lifespan extension. Another study found that cyclin D1 over-expression in primary oral keratinocytes extend the lifespan, whereas the combination of cyclin D1 over-expression and p53 inactivation led to their immortalisation (Opitz et al, 2001). Cyclin D1 forms complexes with CDK4 which subsequently phosphorylates and inactivates retinoblastoma (Rb) growth repression (Connell-Crowley et al, 1997). P16 inhibits cell cycle progression by inhibiting CDK4. It appears that the over-expression of cyclin D1 bypasses p16-TIS resulting in an extension of lifespan. In this instance the p16 mechanism has become redundant and the cells continue to divide until TDS mechanism is activated. This may be why these cells are immortalised by p53 inactivation.

This telomere-independent mechanism may be the primary trigger of cellular senescence in mice and rats. Mice and rats have telomeres which are 5-10 times longer than those of human cells (Shay and Wright, 2001). Despite this increase in length, rodent cells engineered to lack telomerase show telomere shortening with no effect on replicative potential. These cells senesce in the presence of long telomeres. Since mouse and rat cells repair DNA damage far less efficiently than do human cells, it has been suggested that such damage may be the trigger for p16 induction.

Replicative senescence at the molecular level in vitro

2008-04-03T14:52:00.005+01:00

Telomere-dependent senescence (TDS)

Telomere attrition is thought to be the predominant mechanism which ultimately leads to a cell becoming senescent, caused by the inability of polymerase to completely replicate DNA at the 5’ end. The presence of a short telomere is thought to disrupt a protective telomeric structure, exposing loose ends of DNA which trigger a DNA damage response and consequently causes a cell to enter cellular senescence.

Human telomeres consist of short tandem GC-rich repeats (TTAGGG/AATCCC). These telomeric repeats are predominantly double-stranded with a 3’ single-stranded overhang terminating with a G-rich tail (Collins, 2000). The proposed mechanism by which telomeres and their associated proteins protect the chromosome ends is by creating large terminal loops known as a t-loop (Greider, 1999). In this structure, the double-stranded telomeric DNA is looped around and the 3’ single-strand overhang invades the duplex of telomeric repeats and forms a displacement loop (D-loop) structure is important for maintaining chromosomal stability. Without this protection, it is thought that the ends of chromosomes would become exposed, resembling double-strand breaks which can trigger cell-cycle arrest and senescence (Shore, 2001).

Telomeres can be elongated by adding telomeric repeats to the 3’ end of DNA. This can be carried out by telomerase, an enzyme which consists of an RNA molecule and a catalytic component known as hTERT. Telomerase is a reverse transcriptase which uses its RNA component as a template to reverse transcribes DNA back to the ends of chromosomes. Telomerase activity is repressed in most somatic tissue and reactivated in ~90% of human cancers (Artandi, 2006). Introduction of telomerase into normal somatic cells has been shown to extend replicative life-span (Bodnar et al, 1998) and not induce changes associated with a malignant phenotype (Jiang et al, 1999). Since telomerase is not present in somatic cells, they progressively get shorter.

The majority of short-telomere growth arrested cells is thought to occur in the G1 phase of the cell cycle, a process regulated by proteins such as p21. The appearance of DNA damage results in the activation of p53, a transcription factor whose activation results numerous downstream responses including the up-regulation of p21 and activation of DNA repair proteins. P21 is a cyclin dependent kinase (CDK) inhibitor which halts cell cycle progression by directly interacting and inhibiting cyclin-CDK complexes and consequently preventing the phosphorylation of Rb.

Experiments in which p21 was inactivated within senescent cells resulted in the ability of these cells to re-enter the S phase of the cell cycle, but were still unable to divide (Ma et al, 1999). It was concluded that p21 plays an important role in the maintenance of senescence and in the inhibition of S-phase progression, but inhibition of p21 is insufficient to permit cells to complete the cell cycle. Since p21 expression has been shown to be dependent upon p53, the impact of p53 inactivation on cell cycle progression was also investigated (Bond et al, 1999). Fibroblasts lacking p53 function bypassed replicative senescence and continued to proliferate for another 20 CPD before entering growth arrest. The point at which growth arrest occurs due to replicative senescence is also known as M1, whereas the second growth arrest point if replicative senescence is bypassed is known as M2 or crisis. Cells which enter crisis result in apoptosis rather than senescence. This suggests that when a cell becomes senescent at M1 it may not be due to the disruption of the telomere-end structure. Since there appears to be a reserved replicative capacity, it may only be at M2 that the telomere end-structure becomes disrupted, and this may be the reason why apoptosis is common at this point. The idea that a cell becomes senescent long before the telomere-end structure is disrupted suggests that telomere length may not be the trigger for replicative senescence and instead is due to another mechanism.

One such mechanism may be alterations or exposure of the telomere 3’ single-stranded overhang. One study reported that exposure of telomere 3’ overhang sequences induces senescence (Li et al, 2003). In this study, cells were exposed to oligonucleotides homologous to the telomere 3’ overhang sequence TTAGGG for 1 week. As a result, a senescent phenotype in cultured fibroblasts was observed. The team concluded that exposure the 3’ overhang by t-loop disruption or possibly DNA damage leads replicative senescence. Telomere shortening may result in the exposure of the 3’ overhangs, but the telomere end-structure is still maintained efficiently to prevent complete disruption. Interestingly, this 3’ overhang has been found to be eroded at replicative senescence indicating it as a possible trigger (Stewart et al, 2003). It was indicated that overhang erosion is the result of continuous cell division and not a consequence of senescence. Therefore it was concluded that this alteration in telomere structure and not overall telomere length serves as a trigger for replicative senescence.

Replicative senescence at the molecular level in vitro

2008-03-13T14:18:00.003Z

Basic eukaryotic cell cycle

The cell cycle of most cells consists of four coordinated processes: cell growth, DNA replication, distribution of the duplicated chromosomes to daughter cells and cell division. The completion of all these processes takes approximately 24 hours. The cell cycle is divided into two basic parts: mitosis and interphase. Mitosis (or M phase) occurs at the end of the cell cycle and lasts only about an hour. During mitosis, the separation of daughter chromosomes occurs followed by cytokinesis (cell division). The majority of the cell cycle is spent in interphase-the period between mitosis. During interphase both cell growth and DNA replication occur. The cell grows at a steady rate throughout interphase with most cells doubling in size between one mitosis and the next. DNA synthesis however occurs only in a defined phase of interphase. Within interphase there are three distinct phases. Following M phase is the G1 phase, S phase and G2 phase (Figure 1). During G1, the cell is metabolically active and continuously grows, but does not replicate its DNA. During S phase the complete genetic material of the cell must be duplicated. In G2, the cell continues to grow and proteins are synthesised ready for mitosis. Cells in G1, which are not ready to progress through the cell cycle, enter a resting stage known as G0 or quiescence. Most eukaryotic cells spend the majority of their time in a quiescent state.

Throughout the cell cycle there are a number of checkpoints which regulate cell progression from one phase to another. There is a G1 checkpoint that ensures everything is ready for DNA synthesis, a G2 checkpoint to determine whether the cell can proceed to M phase and a checkpoint within M phase to ensure the cell is ready to complete cell division.

Entry into each of the phases of the cell cycle are controlled by two classes of molecules, cyclins and cyclin dependent kinases (CDKs). Cyclins form the regulatory subunits and CDKs the catalytic subunits of an activated heterodimer; cyclins have no catalytic activity and CDKs are inactive in the absence of a partner cyclin. When activated by a bound cyclin, CDKs perform a biochemical reaction called phosphorylation that activates or inactivates target proteins to orchestrate coordinated entry into the next phase of the cell cycle. Different cyclin-CDK combinations determine which downstream proteins are targeted.

For example, during the latter stages of the G1 phase cyclin D1 forms a complex with CDK4, which subsequently phosphorylates and inactivates retinoblastoma (Rb) growth repression (Connell-Crowley et al, 1997). Conversely, growth arrest caused by DNA damage for example is the result of an up-regulation of CDK inhibitors such as p21 and p16 which bind to and inhibit the activity of CDK thereby preventing the phosphorylation of Rb (Aprelikova et al, 1995).

Figure 1: Basic overview of the eukaryotic cell cycle, showing each of the different phases

Replicative capacity of cells from disease states

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The gradual appearance of senescent cells may contribute to the development of age-related disease. However, the presence of disease by other mechanisms may result in accelerated senescence. Disease may cause tissue damage which leads to cellular turnover for the purpose of replacing lost cells. This exhausts the replicative capacity of the cells and accelerates the appearance of senescent cells. For example Goldstein and co-workers (1978) looked at the replicative lifespan of fibroblasts from normal, prediabetic, diabetic donors. Diabetes mellitus is a common genetically determined disorder associated with reduced life expectancy. This study confirmed earlier findings that there is an inverse correlation between donor age and replicative lifespan, but emphasised the importance of physiological state of the donors. Normal cell strains showed significantly better growth capacity than diabetic and prediabetic cells. The results indicated that with an increasing predisposition to diabetes, there is a progressive decrease in replicative capacity.

Another group investigating atherosclerosis took vascular smooth muscle cells (VSMC) from human atherosclerotic plaques and grew them in culture (Bennett et al, 1998). Results showed that VSMCs taken from plaques have lower rates of proliferation and underwent senescence earlier than cells derived from normal vessels.

A more recent study looked at the replicative capacity of osteoblasts in Rheumatoid arthritis (RA) compared with Osteoarthritis (OA) (Yudoh et al, 2000). The results indicated that the replicative capacity of osteoblasts decreased gradually with donor age and this decrease was higher in RA patients than with OA patients at any donor age. They also reported an increase in senescent osteoblastic cells with age in both groups in which the rate of expression of senescent cells was higher in RA patients than with age-matched OA patients.

Tesco et al (1993) looked at the replicative capacity of fibroblasts in patients with familial Alzheimer’s disease (FAD) to examine whether features compatible with a systemic premature aging were present. Data showed that there was no significant difference in replicative capacity of fibroblasts between FAD patients and controls. This is not a surprising result, since the fibroblasts studied are unrelated to the development of FAD and if features of premature ageing were present they would have most likely manifested themselves as other diseases other than just Alzheimer’s. For example, Werner’s syndrome is a premature ageing disorder which displays a multitude of age-related afflictions including diabetes and heart disease (Kipling and Faragher, 1997). When fibroblasts were taken from patients with Werner’s syndrome and grown in culture, the number of population doublings achieved was smaller compared with normal cells of a similar chronological age (Martin et al, 1970)

These studies suggest that disease is an important factor contributing to the exhaustion of the replicative capacity of cells. However, it is possible that some diseases arise as a result of the gradual increase in senescent cells with time. It is also possible that unknown factors result in accelerated senescence, which subsequently manifests itself as a biological impairment or disease.

Factors, other than disease, which may contribute to cellular injury and cell loss, may be environmental such as UV radiation, chemical damage from smoking and foods, and normal biological damage from general wear and tear.

Relationship between replicative capacity and organismal ageing

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Leonard Hayflick was the first to propose that the senescence of normal cells may contribute to the organismal ageing. Investigations into this proposal started by comparing the replicative potential of cells, usually fibroblasts, extracted from individuals at various ages.

The first of these studies showed an inverse relationship between donor age and the number of population doublings achieved in vitro (Martin et al, 1970). This study looked at the replicative lifespan of fibroblasts taken from 100 subjects with an age range from foetal to 90 years. These cells were cultured and the number of population doublings before entering senescence was recorded. The results showed that the replicative potential decreased as donor age increased. A later study showed similar results (Schneider, 1979). This study looked at the ability of fibroblasts taken from young (20-35 years) and old (65+ years) to proliferate in culture. It was reported that cell cultures from old human donors have a reduction in their proliferative capacity. A more recent study looked at the replicative capacity of human adrenocortical cells to proliferate as a function of donor age (Yang et al, 2001). Again, it was found that younger cells have a higher proliferative capability than the old. In this instance, population doubling fell from 50 for foetal cells to almost a total lack of division in culture from older cells.

To investigate the possible link between replicative lifespan and organismal ageing, a few studies compared replicative capacity with longevity in animals. One such study investigated the relationship between longevity of eight mammalian species (mouse, rat, rat-kangaroo, mink, rabbit, bat, horse and human) and the lifespan of normal fibroblasts in vitro (Röhme, 1981). It was reported that there was a direct relationship found between the longevity of the eight mammalian species and the replicative capacity of their cultured fibroblasts. A much later, but similar study, compared animal life spans and in vitro replicative capacity of skin fibroblasts in groupings of small, middle, large, and very large breeds of dogs of specific ages (Li et al, 1996). It was found that the life spans were inversely correlated to the frame sizes of the breeds. It was shown that all the small breeds studied have a longer life span than that of the large breeds. The replicative capacity of fibroblasts from the large dogs (Great Dane and Irish Wolfhound) was significantly decreased compared with that of the small dogs. The reasoning behind these observations may again be due to varying degrees of cell turnover between the species. Large dogs consist of more cells than small dogs and as a result more cell turnover was initially required in their development compared to small dogs. This increase in cell turnover would subsequently lead to a decrease in replicative potential and an increase in the rate of senescent cell formation.

Interestingly, a recent study looked at the replicative capacity of 124 skin fibroblast cell lines from donors of different ages which were medically examined and declared “healthy” (Cristofalo et al, 1998). Healthy people were used specifically as previous studies, discussed later, have shown that disease states may accelerate the reduction in replicative capacity. Results indicated that there was no significant correlation between the replicative capacity of the cell lines and donor age. In the same study, a comparison of multiple cell lines established from the same donors of different ages also failed to show any significant differences. It was concluded that the replicative capacity of fibroblasts in vitro does not correlate with donor age. However, differences in replicative capacity with age may only be observed as a result of increased cell turnover in response to disease and cellular injury. Therefore, a healthy old person who has had little or no cellular injuries or disease would have had little cell turnover and therefore have cells which may have a replicative capacity similar to someone much younger. Thus, this study supports the notion that replicative capacity is an indicator of biological age.

Replicative capacity of tissues from normal human populations

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The maximum replicative potential for mitotic cells varies between different cell types. Some cell types, such as endothelial cells, may have a maximum replicative capacity around 30 cPD (cumulative population doublings) while other cell types such as embryonic fibroblasts may have a maximum replicative capacity of 100 cPD. For example, one early study looked at the replicative capacities of several different tissue types (skeletal muscle, bone marrow spicules and mesial of the midupper arm) taken from donors of the same age (Martin et al, 1970). It was found that the replicative capacity of these tissues, despite being taken from the same individual, displayed variation in their replicative capacity. Cultures derived from skin fibroblasts achieved the greatest number of population doublings, bone marrow spicules the least and skeletal muscle giving intermediate results. There are a number of explanations for these observations. The first is that all cells do have the same replicative capacity, but the replicative history (rate of cell turnover) of each tissue at the time of extraction is so different that such variation is observed. Some tissues may have undergone a higher rate of cellular turnover than others, thereby exhausting its replicative capacity earlier. The second is that the replicative history of each tissue is similar, but it is the length of the telomeres between tissues that differs. Some tissues may senesce sooner than others because they started out with shorter telomeres. It is unlikely that these explanations alone are correct. A combination of the two is the most likely cause for such variation in replicative capacity. Tissues differ in both their replicative history and replicative capacities.

The results also show that the replicative capacity of the same tissues between individuals of the same age also differs. This difference may again be due to the same differences which effect proliferative variability between different tissues of the same individual. For example, one individual may have a shorter replicative capacity in a particular tissue than another of the same age due to increases in cell turnover, maybe in response to disease or injury, or maybe differences in initial telomere lengths. Cultured human embryonic fibroblasts were found to senesce at 50±10 cPD (Hayflick and Moorehead, 1961). This meant that some cultures were senescent only after 40 cPD while others at 60 cPD. These differences in replicative lifespan may be a consequence of the stochastic mechanism which triggers a cell to senesce. Therefore, the difference in replicative capacities of the same tissues between individuals of the same age may also be due to the stochastic events which govern a cell becoming senescent. Thus, the replicative capacity of a tissue measures biological age and not chronological age. Unfortunately there have been few studies looking at the replicative capacity of different tissues from the same individuals. This would have given a better insight into the relationship between chronological and biological age.

Replicative senescence at the cellular level in vitro

2008-02-15T12:52:00.004Z

Historical overview

Until the middle of the 20th century, it was widely held that normal mitotic tissue could not age in a degenerative sense because it had an indefinite capacity to proliferate. This view had developed for two reasons. Firstly, the Nobel laureate Alexis Carrel appeared to have demonstrated the long-term cultivation of normal chick fibroblasts for periods considerably in excess of the lifetime of the animal (Parker, 1938; Witkowski, 1990). Secondly, the technical difficulties associated with tissue culture techniques until the 1950s rendered the duplication of Carrel’s studies very difficult for all but a few highly specialized laboratories (Parker, 1938). Ageing was not the primary research interest of these centres. Thus, it was not until a series of classic experiments by Hayflick & Moorhead in the early 1960s which demonstrated that cultures of normal human fibroblasts did not have an infinite capacity to expand, brought about the idea of intrinsically immortal mitotic tissue into question. Their work demonstrated that, after a finite period of growth, cultures of normal human fibroblasts became completely composed of viable but non-dividing cells (Hayflick & Moorhead 1961; Hayflick 1965). These initial observations have been reproduced in hundreds of studies, and since that time virtually all human mitotic cell types subjected to rigorous study have been shown to undergo this cellular senescence in culture.

Dynamics of normal cell populations.

The early observation by Hayflick and Moorehead (1961) that cultured cells have a maximum limit on the number of divisions before entering senescence lead to the assumption that all cells in a given culture divide roughly the same number of times before entering senescence. Hayflick considered the senescence of cultures was marked by three distinct phases. In Phase 1 the initial culture, was considered to terminate with the formation of the first confluent sheet of cells. Phase 2 was characterised by vigorous growth requiring repeated subculture. In phase 3, the senescence of the culture was characterised by the cessation of mitosis. In this model it was assumed that cultures were composed of a homogenous population of cells which were either all growing (Phase 1 or 2) or all non-growing (Phase 3) and that failure to grow was due to cell death (Kalashnik et al, 2000). The notion that senescence was cell death was soon disproved with the demonstration that RNA synthesis occurred in these cells (Macieira-Coelho et al, 1966). Evidence against the idea that cultures were composed of homogenous populations was provided by a number of different studies. Cristofalo and Scharf (1973) demonstrated the presence of senescent cells in early passage cultures using long pulse-labelling experiments on embryonic fibroblasts. 3H-thymidine labels those cells which have entered S-phase (dividing cells), and since senescence cells are halted in G1 they cannot enter S-phase, and so the percentage of unlabelled cells can be calculated. It was shown that senescent cells are present in early passage cultures and that the percentage of senescent cells gradually increases with each serial passage of the culture. This observation was explained by experiments demonstrating that cultured fibroblasts are composed of cells which display variation in proliferative potential (Smith and Whitney, 1980). Related experiments also showed that two cells arising from a single mitosis differed in their ability to proliferate by as many as eight doublings (Jones et al, 1985). Using the miniclone technique the replicative capacity of individual cells growing in bulk culture can be measured as well as the sizes of colonies generated by dividing cells (Ponton et al, 1983). Results showed that the percentage of glial cells capable of dividing gradually decreases with every new passage. This data is based on the broad distribution of colony sizes which showed a shift from many large colonies to more small colonies as population doublings increased.

Modern techniques for measuring the dynamics of normal cell populations involve measuring not only the senescent fraction of cells, but also the proliferating and apoptotic fraction. The most commonly used method to visualise senescent cells both in culture and in vivo is the senescence-associated beta-galactosidase assay (SA-β-Gal) (Dimri et al, 1995). Although this is a safer method than using 3H-thymidine labels, its robustness as a biomarker is questionable since the assay is dependent upon lysosomal mass (and cell size) rather than growth state (Lee et al, 2006). Cellular proliferation markers such as bromodeoxyuridine (BrdU) and Ki67 are commonly used to label and calculate the proliferating fraction. For measuring the apoptotic fraction, terminal transferase dUTP nick end labelling (TUNEL) is a commonly used method. This assay can detect DNA fragmentation that results from apoptotic signaling cascades.

An example of these methods being used for determining the growth dynamics of human umbilical vein endothelial cells (HUVEC) can be observed in a paper by Kalashnik et al (2000). Results show a gradual decline in the growth fraction as measured by Ki67, an increase in the senescent fraction and the apoptotic fraction remaining unchanged with each serial passage.

These findings thus suggest that the mechanisms resulting in cellular senescence is a stochastic process. As the proliferative capacity of cells declines with age or with increasing population doublings, the mechanism leading to cellular senescence is one in which these stochastic events gradually increases.

Overview of mechanisms of ageing

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Ageing is a multi-causal process with some ageing mechanisms being more predominant in some tissues than in others. This is due to the difference in the biological make-up of tissues which makes them more or less predisposed to a particular ageing mechanism. Since tissues are normally composed of a mixture of mitotic cells, post-mitotic cells and long-lived proteins that are functionally inter-related, there is going to be considerable overlap in the ageing mechanism within tissues. An alteration in one tissue component will likely have a direct impact on another. The outcome of such changes is going to be different from tissue to tissue due to impairment of specific structure-function relationships.

Mitotic cells

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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

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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

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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

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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

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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

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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

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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

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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.

Why do we Age?

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Early theories on this subject (Medawar, 1952, Haldane, 1941) tried to understand how something as negative as ageing could have been positively selected for in evolution, especially since most animals in the wild do not live to old age. Only captive animals that are protected from predation, starvation or physical trauma, live to and die from old age. To give an explanation for this problem, Medawar hypothesised that there is a declining force of natural selection with age. Harmful genetic events that are expressed prior to sexual maturity of an individual will be strongly selected against, whereas the expression of such changes at later stages will not be subject to such negative selection. Later, the term antagonistic pleiotropy was contributed to the topic of evolutionary ageing (Williams, 1957). Antagonistic pleiotropy refines the ideas set by Medawar by suggesting that late-acting deleterious genes may be favoured by selection and be actively accumulated in individuals if they have beneficial effects early in life. Such beneficial effects may include the enhancement of an individual’s ability to survive until the reproductive period and/or to carry out reproductive activities in a successful manner.

Another explanation for the conflict between reproduction and longevity came much later with the Disposable Soma theory (Kirkwood, 1977). Unlike the antagonistic pleiotropy theory, the Disposable Soma theory does not explicitly implicate genes as being involved in the ageing process. It instead focuses on the distribution of precious metabolic resources for either increasing reproductive capacity or somatic cell maintenance (DNA repair, protein turnover and antioxidant defences etc). Therefore, organisms have evolved in such a way that the amount of energy invested in maintaining the somatic tissue is sufficient to keep the individual alive long enough to reproduce, but less than what is required to keep it alive indefinitely.

Definition of normal Ageing

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Ageing, the process of growing old, is defined as the gradual biological impairment of normal function, probably as a result of changes made to cells (mitotic cells, such as fibroblasts and post-mitotic cells, such as neurons) and structural components (such as bone and muscle). These changes would consequently have a direct impact on the functional ability of organs (such as the heart, kidney and lungs), biological systems (such as the nervous, digestive and reproductive system) and ultimately the organism as a whole.

The following is a description setting out five criteria’s for ageing, as proposed by Strehler (1962).

Cumulative: Effects of ageing increase with time.
Universal: All members of a species display signs of ageing.
Progressive: Ageing is a series of gradual changes.
Intrinsic: Changes would take place even in a “perfect” environment.
Deleterious: Changes which occur compromise normal biological functions.

Like ageing, disease is also defined as an impairment of normal function within a living organism. Since the ageing process leads to biological impairment, it would not therefore be a surprise if some of these age-associated changes manifest themselves as disease. Evidence providing a link between ageing mechanisms and age-related disease development/progression is gradually increasing (discussed at a later date).

Problem of Ageing

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Increasing life expectancy is a positive development, but is not without its own problems. The prevention or elimination of causes of death in early life has resulted in a population consisting of an increasing proportion of elderly people. The United Nations populations division estimated that the number of persons aged 60 years and older in 1999 was nearly 600 million worldwide and was projected to reach about 2 billion by 2050 (UN,1999). This means that by the year 2050, the population of older people will be larger than the population of children for the first time in history. In the UK alone it has been estimated that the number of people over the age of 65 is expected to rise by 81 percent over the next five decades (Government Actuary’s Department; www.gad.gov.uk).

The ageing process is associated with an increasing rate of morbidity, the period of time spent sick before recovery or death. Thus, since the elderly population is increasing, more and more people are going to require care. Therefore, to keep pace with demographic change, the number of places taken in residential care homes, nursing homes and hospitals would have to rise. More money is going to be required to keep up with the needs of a changing population, especially in healthcare. It has been estimated that total UK spending on long-term care would rise from £12.9 billion in 2000 to around £53.9 billion by 2051 (London School of Economics; www.lse.ac.uk).

Improvements in general health or advances in treatment of disabling illnesses could lead to a reduction in the proportion of older people needing residential and nursing home care. In order to develop new treatments and management strategies to help deal with the consequences of an elderly population, a clear in-depth understanding of the molecular mechanisms which contribute to the human ageing process and the development of age-related diseases is thus required.

Introduction

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During the latter half of the 20th century the average life expectancy throughout the developed world began to increase rapidly. The term “life expectancy” is often confused with “lifespan”, which measures a subtly different parameter. Life expectancy represents the likelihood of surviving to a given age and is often an indicator of the overall health of a country. Increases in life expectancy in developed countries are mainly due to improvements in medicine, public health and nutrition. The primary cause of death in such countries is age-related diseases such as, cardiovascular disease, strokes, infection as result of impaired immune function and numerous cancers. Conversely, life expectancy can fall due to problems such as famine, war, disease and poor health. For example, in regions of Africa and Asia where AIDS is a predominant cause of death, life expectancy is dramatically decreased compared to developed countries and is expected to decrease further if medical intervention is not effective (Logie, 1999). The average life expectancy of a person living in a developed country is about 75 years compared with that of 43 years in some regions of Africa. Lifespan on the other hand is the maximum survival potential as defined by the longest surviving member of a population. For humans, it has been estimated to be between 115 and 120 years. Increases in life expectancy have not resulted in any significant increase in the maximum human lifespan.