VSMC senescence and calcification

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

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

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

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.

Microarray analysis of senescent keratocytes (EK1.Br)

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

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.

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See: Bridging the gap: ageing, pharmacokinetics and pharmacodynamics

Cellular senescence papers: different cell types

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

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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)

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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

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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

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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

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.

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ALSO SEE:

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Phenotypic changes associated with cellular senescence

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DISEASE FOCUS: Atherosclerosis and vascular calcification
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Cellular senescence in disease states

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Cellular senescence, ageing and disease (2009)

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