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Ageing Research

A blog for those interested in learning more about the ageing process, specifically concentrating on cellular senescence and it's impact on age-related tissue dysfunction and disease development/progression.

Updated: 2009-10-13T02:01:23.023+01:00

The need for an effective biomarker of senescent cells

2009-10-08T18:01:43.348+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.

DISEASE FOCUS: Atherosclerosis and vascular calcification

2009-08-10T22:06:27.516+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 cellsThe 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

2008-05-18T13:43:03.264+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

2008-05-18T13:42:41.601+01:00

Inflammation and atherosclerosisAtherosclerosis 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 (Og[...]

DISEASE FOCUS: Atherosclerosis and vascular calcification

2008-05-18T13:42:20.267+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

2008-05-02T22:04:53.373+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 fibrobl[...]

Cellular senescence in disease states

2009-03-31T10:16:59.869+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 mont[...]

Replicative Senescence in vivo

2008-04-29T01:29:23.204+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.

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