2046-2395-2-3 2046-2395 Review <p>A relationship exists between replicative senescence and cardiovascular health</p> Karavassilis E Maria Maria.karavassilis11@imperial.ac.uk Faragher Richard R.G.A.Faragher@brighton.ac.uk

School of Medicine, The Commonwealth Building, The Hammersmith Hospital, Imperial College London, Du Cane Road, W12 0NN, London, UK

School of Pharmacy and Biomolecular Sciences, University of Brighton, Huxley Building, Lewes Road, BN2 4GJ, Brighton, UK

Longevity & Healthspan 2046-2395 2013 2 1 3 http://www.longevityandhealthspan.com/content/2/1/3 10.1186/2046-2395-2-3
23 1 2012 12 11 2012 4 2 2013 2013 Karavassilis and Faragher; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Replicative senescence Vascular endothelial cells Vascular smooth muscle cells Ageing Atherosclerosis Hypertension

Abstract

A growing body of evidence demonstrates that the accumulation of senescent cells is a plausible ageing mechanism. It has been proposed that the senescence of vascular cells plays a causal role in the development of cardiovascular pathologies. A key prediction arising from this hypothesis is that cultures of cells derived from donors with cardiovascular disease will show reduced in vitro replicative capacities compared to those derived from disease-free controls. Accordingly, we carried out a formal review of the relationship among donor age, cardiovascular health status and maximum population doubling level attained in vitro by cultures of vascular smooth muscle and endothelial cells. Data were available to us on a total of 202 independent cell cultures. An inverse relationship was found to exist between replicative capacity and donor age in both endothelial and vascular smooth muscle cells. Cultures derived from donors with cardiovascular disease showed a lower overall replicative potential than age-matched healthy controls. In general the replicative potential at the start of the lifespan was found to be higher in those individuals without disease than those with disease and the difference in average cumulative population doublings (CPDs) in age-matched individuals in the two groups remained roughly constant throughout the lifetime. These results are consistent with the model in which the inherited replicative capacity of vascular cells is a stronger determinant of the onset of cardiovascular disease later in life, than wear-and-tear throughout the life course.

Review

Introduction

The purpose of this article

A detailed understanding of the mechanisms underlying cardiovascular disease has important implications for the reduction of mortality and morbidity in the aged population. For largely historical reasons, the majority of studies of replicative senescence have focused on human fibroblasts, as it was in these cell types that the phenomenon of cellular senescence was first reported. Unfortunately, this has produced a distortion of the evidence base, which renders a detailed understanding on the relationship replicative senescence and health status problematic. This is because very few age-associated pathologies are directly attributable to the dermal layer of the skin (or other fibroblastoid tissue layers, such as the corneal stroma).

However, a number of cell types exist in which a direct link between the senescent phenotype and age-associated disease is potentially easier to interrogate. Endothelial cell (EC) and vascular smooth muscle cell (VSMC) senescence in particular has been linked to the development of cardiovascular disease, specifically atherosclerosis 123. The senescent phenotype has been proposed to result in an impaired ability to replace damaged or lost cells or to produce altered tissue microenvironments within the vessel 4. Accordingly we reviewed the available data on proliferative capacity, donor age and cardiovascular disease status with regard to these cell types, in order to produce a meta-dataset spanning a range of age groups and cardiovascular disease states, allowing provisional conclusions to be drawn.

The cell hypothesis of ageing

The hypothesis that the progressive accumulation of senescent cells with tissue turnover during life plays a causal role in ageing was first proposed more than 40 years ago and has gained increasing credibility in recent years 5. Classic studies in this area include that of Martin et al.6, who reported a reduction of approximately two population doublings per decade in fibroblasts derived from individuals aged from new-born to a hundred years, and that of Schneider and Mitsui 7, who demonstrated a reduced average fibroblast migration rate, lower replication rate and saturation density at confluence, in fibroblast cultures derived from older individuals compared to their younger counterparts.

However, several studies using fibroblast cultures have demonstrated no statistically significant decline in replicative potential with age 89. One explanation for lack of correlation is the exclusive selection of healthy donors. Because senescent cells are highly likely to be causal agents of age-related disease 5, it follows that these may be absent in a study population comprising only healthy donors.

Maier and Westendorp (2008) 10 proposed that the relationship between senescence and ageing is attributable to publication bias, and is absent in studies comprising large sample populations. However, this work contains questionable methodological aspects, the major constraint being the use of data derived from the fibroblast literature. Skin samples are simply not directly reflective of the pathological mechanism underlying Alzheimer’s disease.

There are literature reports where the proliferative capacity of fibroblasts from centenarians is not significantly reduced compared to those from younger donors 11. This may reflect the fact that fibroblast replicative capacity in culture is not representative of the key organ systems in vivo.

The notion that senescence is a causal process driving at least some aspects of ageing is undeniable, as senescent cells have been found to be present and accumulate in tissue in vivo as a function of organismal age 1213. Among the best evidence of this is provided by a study where the number of mitotic cells in the proliferative region of murine lens epithelium was seen to decline with the age of the organism, with a concurrent increase in senescent cells 14.

The presence of senescence-associated markers at sites of pathology supports the relationship between replicative senescence and age-related disease. Using senescence-associated beta-galactosidase, senescent endothelial cells have been found to accumulate after repeated balloon endothelial denudation of the rabbit carotid artery 15. It has also been demonstrated that telomeres in the endothelium shorten with age and this is more pronounced in atherosclerosis-prone areas 141516. Moreover, cultures of vascular smooth muscle cells derived from plaques have been reported to show a reduced proliferative capacity 17.

The senescent phenotype of vascular cells is not inconsistent with the potential to cause cardiovascular disease. Senescent endothelial cells in vitro have been found to over-express proteins characteristic of the pro-inflammatory and pro-thrombotic phenotype of the endothelium in human atherosclerosis, including IL-1alpha, ICAM-1 and PAI-1 181920. Burton et al. (2009) demonstrated that key genes known to be up-regulated in atherosclerotic plaques are also highly up-regulated in senescent VSMCs. Most importantly, it was found that senescent VSMCs adopt a phenotype which contributes to the pathogenesis of vascular calcification, characterized by the expression of genes associated with vascular calcification, namely matrix Gla protein, bone morphogenetic protein-2, osteoprotegerin, osteopontin and decorin 21.

Hypothesis

The relationship between senescence and disease state throughout the lifetime can be modeled in three ways. In the first hypothesis, all individuals are expected to show similar inherited replicative capacities at the start of their lifespan. In subjects without cardiovascular disease, senescence is assumed to be the consequence of lifelong reparative cell divisions with advancing age 222324. In subjects with cardiovascular disease, senescence may be thought to represent an acceleration of the biological ageing process, triggered by exposure to mitotic stress, oxidative stress or DNA damage, independent of chronological age 252627. Those more prone to developing disease are, therefore, expected to exhaust their replicative potential at an accelerated rate compared to healthy individuals. The expected graphical relationship shows the trend lines of diseased and healthy individuals originating on the same point on the Y-axis, from which they diverge, with the line representing diseased individuals showing a steeper slope (Figure 1).

<p>Figure 1</p>

Hypothesis 1: diseased and healthy individuals start with similar replicative capacities

Hypothesis 1: diseased and healthy individuals start with similar replicative capacities. Replicative capacity is exhausted at a faster rate in diseased individuals.

It may otherwise be the case that replicative capacity always declines consistently with chronological age, regardless of health status (Figure 2). In this case, the expected graphical relationship should show that both trend lines coincide throughout the organism’s lifespan.

<p>Figure 2</p>

Hypothesis 2: diseased and healthy individuals coincide in terms of replicative capacity throughout lifespan.

Hypothesis 2: diseased and healthy individuals coincide in terms of replicative capacity throughout lifespan.

An alternative hypothesis is that individuals differ in inherited replicative capacity at the start of their lifespan, but exhaust their replicative potential at a similar rate. Those more prone to developing disease are expected to start off with a lower replicative capacity (Figure 3). The expected graphical relationship should show the trend line corresponding to diseased individuals to start out at a lower point on the y-axis compared to non-diseased individuals, after which point the trend lines decline at similar rates, appearing to be parallel.

<p>Figure 3</p>

Hypothesis 3: diseased and healthy individuals start off with different replicative capacities.

Hypothesis 3: diseased and healthy individuals start off with different replicative capacities. Replicative capacity is exhausted at similar rates.

Individual differences in replicative capacity may be attributed to variations of inherited telomere lengths, as has been suggested in several studies. In one report, blood telomere lengths were found to range from 5.10 kb in young men from Italy to 18.64 kb in young men from Belgium, showing a greater than three-fold difference across populations 28. In another study, individuals of African-ancestry in the USA tended to show 10% longer blood telomere lengths compared to those of European ancestry 29. The large variation of telomere lengths observed in adults was suggested to relate to adaptive evolution, differences in early life experience or growth 30, exposure to stress throughout the lifespan 31 and, most pertinently in this case, varying paternal ages at reproduction. Kimura et al.32 investigated the relationship between telomere length in sperm from young (30 years) and older (50+ years) donors with mean leukocyte telomere length in offspring (in adult ages); they demonstrated that a subset of sperm from older men had elongated telomeres, which corresponded to a positive relationship between paternal age at reproduction and offspring telomere length. The variation of telomere length in embryo-derived cells appears not to have been studied in detail.

Certainly, vascular cells with inherited short telomeres that are also exposed to considerable amounts of stress throughout the life course should show a much lower replicative potential compared to healthy individuals. The most likely hypothesis is, therefore, based on the notion that most disease states are multi-factorial in nature. When combining data from many individuals, it is expected that the relationship between replicative capacity and ageing in diseased individuals will show an overall lower cumulative population doubling (CPD) potential compared to healthy controls, as well as an accelerated rate of decrease of CPD.

Materials and methods

Data mining

We mined the available peer-reviewed literature (principally using PubMed and relevant electronic journals directly). Combinations of search terms used included: ‘endothelium’, ‘vascular smooth muscle cells’, ‘senescence’, ‘cumulative population doublings’, ‘in vitro’, ‘passage’ and ‘donor age’. The search covered over 10,000 research articles. When available, the following information was obtained: age of donor, replicative capacity in vitro, vessel of origin, gender, incidence of cardiovascular-related disease and definition of replicative senescence. Articles were excluded if replicative capacity in vitro was recorded in such a way that CPD could not be accurately deduced. Ultimately, 12 research articles were used, published between 1978 and 2008.

Replicative capacity and health status

Studies were included in this review if donor age and replicative capacity of EC or VSMC were recorded. If not given, the CPD value was extrapolated from maximum passages achieved (given the cell culture split ratio) or approximated from scatter plots. If health status was not mentioned, it was assumed that the donors did not suffer any cardiovascular-related diseases.

Statistical analysis

To assess the strength of the correlation between in vitro replicative capacity and age of donor, regression analyses were carried out for each cell type, as well as sample populations with/without vascular–related diseases separately. For endothelial cells, graphs including all data points as well as graphs excluding human umbilical vein endothelial cells (HUVECs) are shown, as HUVEC samples show a very large scatter, which affected the trend lines. The mean decade CPD was plotted against donor decade age, to balance out any effects from possible confounding variables.

The mean rate of change of CPD with respect to age (change in CPD per year) was calculated for the graphs of: ‘all CPD values versus donor age’, ‘mean CPD versus donor age decade’ and for the graphs of ‘CPD versus donor age’ in those with or without vascular-related diseases shown separately.

To assess the contribution of confounding factors, scatter plots were drawn to visualize clustering of data points, per vessel type and per gender.

T-tests were performed to assess whether the difference in mean CPD for adjacent decades as well as decades at either extreme, could be attributed to chance. A T-test was also carried out comparing CPD of male versus female donors for each cell type.

Results

Replicative capacity in vitro versus age of donor

The linear regression line shows a clear inverse relationship between replicative capacity in vitro and donor age, indicating a negative correlation for VSMC and EC. For both cell types, it is evident from the graphs that in the 50+ age group there are a larger proportion of points with low CPD values, that is, below 10 CPD in VSMCs and below 20 CPD in ECs.

When the decade mean CPD is plotted against donor age, a negative correlation is seen for both cell types, with R = −0.866 for VSMC (Figure 4), R = −0.733 for EC (Figure 5).

<p>Figure 4</p>

Relationship between the decade mean CPDs and donor age decade in vascular smooth muscle cells.

Relationship between the decade mean CPDs and donor age decade in vascular smooth muscle cells.

<p>Figure 5</p>

Relationship between the decade mean CPDs and donor age decade in endothelial cells.

Relationship between the decade mean CPDs and donor age decade in endothelial cells.

T-tests were carried out between datasets for adjacent decades. The P-values for the comparisons between adjacent decades in both cell types (with the exception of decades 4 and 5 in EC), indicate that one cannot say with certainty that the observed difference for adjacent decades cannot be attributed to chance (Table 1). The P-values comparing the first and eighth decades indicate that the differences are highly significant. The same conclusion was drawn when comparing the first and last two and three decades in VSMC and EC respectively. Thus, based on the available dataset, although it is not strongly evidenced that small age differences show a decrease in CPDs, a large age difference is clearly associated with a substantial decrease in CPD.

<p>Table 1</p>

Cell type

Decades being compared

P-value

T-test interpretation

VSMC

1, 2

0.734

No Sig. Diff.

VSMC

2, 3

0.386

No Sig. Diff.

VSMC

3, 4

0.393

No Sig. Diff.

VSMC

4, 5

0.764

No Sig. Diff.

VSMC

5, 6

0.856

No Sig. Diff.

VSMC

6, 7

0.597

No Sig. Diff.

VSMC

7, 8

0.638

No Sig. Diff.

VSMC

1, 8

3.47E-04

Sig. Diff.

VSMC

1+2, 7+8

1.43E-05

Sig. Diff.

EC (all)

1, 2

0.0205

Sig. Diff.

EC (all)

2, 3

0.715

No Sig. Diff.

EC (all)

3, 4

0.947

No Sig. Diff.

EC (all)

4, 5

0.0235

Sig. Diff.

EC (all)

5, 6

0.444

No Sig. Diff.

EC (all)

6, 7

0.838

No Sig. Diff.

EC (all)

7, 8

0.887

No Sig. Diff.

EC (all)

8, 9

0.635

No Sig. Diff.

EC (all)

1, 8

4.04E-10

Sig. Diff.

EC (all)

1+2+3, 7+8+9

5.46E-12

Sig. Diff.

EC (excluding HUVECs)

2+3, 7+8+9

1.98E-03

Sig. Diff.

Table showing cell type, decades being compared, P-values and T-test interpretation

The mean rate of change in CPD with respect to age was also evaluated by calculating the slope of the linear regression line (Table 2). Table 3 shows the mean rate of change in CPD with respect to age, per publication. One can see that VSMC shows a lower rate of decrease per year (−0.19) compared to EC (−0.44) and similar rate of decrease when excluding HUVECs (−0.15). Looking at the mean decade CPD with respect to age decade, both cell types gave the same value (−0.19) of rate of change.

<p>Table 2</p>

Cell type

Variables

Change in CPD per year

EC

CPD values -vs-donor age (all)

−0.44

EC

CPD values-vs-donor age (excluding HUVECs)

−0.15

EC

Mean CPD values-vs-donor age decade (all)

−0.19

EC

Mean CPD values-vs-donor age decade (excluding HUVECs)

−0.19

VSMC

CPD values -vs-donor age

−0.19

VSMC

Mean CPD values-vs-donor age decade

−0.19

EC

CPD -vs- donor age in donors with cardiovascular-related diseases (all)

−0.14

EC

CPD -vs- donor age in donors without cardiovascular-related diseases (all)

−0.47

EC

CPD -vs- donor age in donors without cardiovascular-related diseases (excluding HUVECs)

−0.16

VSMC

CPD -vs- donor age in donors with cardiovascular-related diseases

−0.17

VSMC

CPD -vs- donor age in donors without cardiovascular-related diseases

−0.2

Table showing cell type, variables and change in cumulative population doublings per year

<p>Table 3</p>

Cell type

Variables

Change in CPD per year

No. of samples

VSMC

Eskin (1981) 47

−0.029

64

VSMC

Fukai (1994) 42

−0.168

12

VSMC

Kan (1987) 48

−0.176

4

VSMC

Bierman (1978) 49

−0.03

17

EC

Hoshi (1986) 50

−0.206

8

EC

Glassberg (1982) 51

0

4

EC

Johnson (1992) 52

−0.216

11

EC

Maciag (1981) 53

NA

4

EC

Nobuhiko (1988) 54

NA

2

EC

Vogel (2008) 37

NA

2

EC

Watkins (1993) 41

+0.45

2

EC

Vogel (2007) 55

+0.119

61

VSMC

TOTAL

−0.19

107

EC

TOTAL

−0.44

113

Table showing cell type, publication, change in CPD per year, and number of samples

Replicative capacity in vitro versus age of donor with respect to cardiovascular-related diseases

The data points from donors with cardiovascular-related diseases show a negative correlation with age, with R = −0.511 in VSMCs (Figure 6) and R = −0.24 in EC (Figure 7). The data points from donors without cardiovascular related disease also show a negative but slightly weaker correlation with age R = −0.411 in VSMC (Figure 6) and much stronger correlation with age R = −0.708 in EC (Figure 7) and −0.799 when excluding HUVEC (Figure 8). It is useful to exclude HUVEC data points as they show a very wide scatter. Comparing the trend lines visually, it is clear that on average, CPDs are higher in those without cardiovascular-related disease compared to those with the disease for both cell types (when excluding HUVEC).

<p>Figure 6</p>

Relationship between CPDs and donor age in VSMCs, showing donors with and without cardiovascular-related diseases.

Relationship between CPDs and donor age in VSMCs, showing donors with and without cardiovascular-related diseases.

<p>Figure 7</p>

Relationship between CPDs and donor age in ECs, showing donors with and without cardiovascular-related diseases.

Relationship between CPDs and donor age in ECs, showing donors with and without cardiovascular-related diseases.

<p>Figure 8</p>

Relationship between CPDs and donor age in ECs, showing donors with and without cardiovascular-related diseases, excluding HUVEC data points.

Relationship between CPDs and donor age in ECs, showing donors with and without cardiovascular-related diseases, excluding HUVEC data points.

The T-tests between the sample populations with or without cardiovascular-related diseases show extremely small P-values of 3.89E-11 for VSMC and 1.21E-14 for EC and 1.11E-2 for EC, excluding HUVEC, indicating with a high degree of confidence that the observed difference between the means of the two data sets is not attributed to chance.

The change in CPD with respect to age in those without cardiovascular-related disease (Table 2) shows a slightly lower rate of decrease per year compared to those without cardiovascular-related disease, in both cell types, that is, -0.14 in ECs from donors with cardiovascular-related diseases, -0.47 in ECs without cardiovascular-related diseases, -0.16 in ECs without cardiovascular-related diseases, excluding HUVECs, -0.17 in VSMCs from donors with cardiovascular-related diseases, -0.2 from donors without cardiovascular-related diseases.

Scatter plots were drawn to assess the effects of vessel type and gender on EC and VSMC replicative capacity (Figures 9, 10, 11, 12). Overall, the data-points are seen to be equally spread in both cell types, regardless of gender or vessel type.

<p>Figure 9</p>

Relationship between cumulative population doublings and donor age in vascular smooth muscle cells, per vessel type.

Relationship between cumulative population doublings and donor age in vascular smooth muscle cells, per vessel type.

<p>Figure 10</p>

Relationship between cumulative population doublings and donor age in endothelial cells, per vessel type.

Relationship between cumulative population doublings and donor age in endothelial cells, per vessel type.

<p>Figure 11</p>

Relationship between cumulative population doublings and donor age in vascular smooth muscle cells, per gender.

Relationship between cumulative population doublings and donor age in vascular smooth muscle cells, per gender.

<p>Figure 12</p>

Relationship between cumulative population doublings and donor age in endothelial cells, per gender.

Relationship between cumulative population doublings and donor age in endothelial cells, per gender.

Discussion

Replicative capacity in vitro versus age of donor

For both cell types there are fewer data points for the younger ages of 0 to 30 years, which is to be expected, as there is less availability for vessel biopsies (with the exception of HUVECs). Comparing the graph of the VSMCs with those of the ECs, the observed trends and average CPD values per age are roughly similar between the two cell types.

The data presented here are consistent with previous findings, showing a definite decline in proliferative activity in vitro with donor age. If the mean decade CPD values are used (to balance out the effects of possible confounding factors), the change in CPDs with age of both cell types corresponds to the finding by Martin, et al.6 of approximately two CPDs per decade in fibroblasts. The findings are also strongly consistent with Hayflick’s observations on fibroblasts (1965) 33; ECs derived from human embryos underwent about 48 (mean average) CPDs in vitro, ranging from 18 to 79 CPDs, while ECs derived from adults underwent roughly 20 (mean average) CPDs, ranging from 3 to 30 CPDs (excluding the two extreme outliers). Hayflick and Martin also emphasized the lack of correlation in the proliferative ability of cells derived from individuals of intermediate ages, despite the clear difference in the proliferative ability of cells derived from very old (70+) or very young (0 to 10) donors. The same conclusions were drawn in this study, as demonstrated by the T-test calculations between adjacent and extreme ages. The exception to this is the difference in CPDs between decades 4 and 5 in ECs. The graph shows a very clear and sudden decrease in replicative capacity between the two decades and the P-value shows a significant difference between the two datasets. A reason for this may be that the decline in replicative capacity as a function of age may not occur at a consistent rate, possibly showing an abrupt decrease at certain points in the lifetime. The relationship between in vitro replicative capacity and organismal ageing should, therefore, not be interpreted to be linear or very tight 34.

Replicative capacity in vitro versus age of donor, with respect to cardiovascular-related diseases

Many studies on vascular smooth muscle cells and endothelial cells have reported a relationship between the altered cellular phenotype associated with senescence and the onset of cardiovascular disease 181920213536. In this review, the graphs show a vivid relationship between cardiovascular-related disease and replicative capacity in vascular cells, supported by the T-test calculations. It is clear from the graphs that individuals with cardiovascular-disease show a lower cellular proliferative potential than those without cardiovascular disease. This suggests that disease state is more strongly associated with the onset of senescence than donor age.

Looking at the replicative capacity for HUVECs alone (limited data for embryo-derived VSMCs), the extremely wide scatter ranging from 18 to 79 CPDs already implies a variation in terms of inherited proliferative capacity, possibly attributed to differences in telomere length. Inherited telomere length is, in turn, thought to be affected by paternal age at reproduction 37. If replicative capacity can be considered a heritable trait, it follows that neonates showing inherent low cellular proliferative capacity will be more prone to disease later in life. Since neonates cannot be distinguished on the basis of liability to disease (and because none are yet diagnosed with disease), including these in the graphical relation between disease and replicative capacity distorts the trend; classing all HUVECs as without disease is misleading, as these will show the trait assumed to underlie disease predisposition (low replicative capacity), as do the adult donors with disease.

When HUVECs are excluded, both ECs and VSMCs show that diseased individuals start off with a lower replicative capacity than non-diseased individuals. The difference in average CPDs in age-matched individuals in the two groups is roughly constant throughout the lifetime, that is, an average difference of 7 CPDs in ECs and 20 CPDs in VSMCs. This essentially supports the hypothesis suggesting that individuals differ in terms of inherited cellular replicative capacity from the start of their lifespan, but exhaust this proliferative potential at similar rates. These conclusions are in line with the assumption that inter-individual inherited telomere length varies widely. Substantial variations of telomere lengths have been reported in some studies 293032; however, the focus has been on adult donors. In adults, telomere shortening may occur following reparative cell divisions, exposure to stress or early infection, as well as differences in inherited telomere lengths. This probably accounts for the scattered distribution of data points corresponding to adult donors observed in this analysis. It would be more interesting if variation in telomere length of cells derived from human embryos were investigated in association with paternal age and telomere length in sperm. As far as we know, population variations of telomere length in neonates (that is, HUVECs) and with respect to paternal characteristics, has not yet been studied. Further exploration to elucidate the extent of variation of inherited telomere length and the possible consequences for disease, as initially implied in this review, is necessary. Despite the obvious inclination of these data toward inherited proliferative activity being strongly associated with disease state, the effect of decline in replicative capacity throughout the lifetime (that is, exogenous stress) should not be ignored. Cells from individuals with inherited short telomeres that are also exposed to considerable amounts of stress throughout the life course should undoubtedly show an even greater decrease in replicative capacity compared to healthy individuals.

While there is evidence that telomere shortening controls senescence in endothelial cells 338, it is unclear whether vascular smooth muscle cells undergo telomere-dependent senescence. It has been demonstrated that in VSMCs telomerase reverse transcriptase (TERT) is regulated at the transcriptional level, it is unclear whether over-expression of telomerase can bypass senescence 339. There are limitations in terms of experimental approaches in these studies: each of these experiments involved analysis of pooled colonies, which is unreflective, since replicative capacity of cells in culture reflects the expansive propagation of the longest surviving clone. A more informative approach should involve isolation of as many colonies of vector-infected cells as possible (if possible, approximately n = 15). If all the isolated clones are found to be immortalized, the cell type can be described with a high certainty to have undergone telomerase-induced escape from senescence.

Limitations of this study

A quantitative relationship between proliferative capacity and age cannot be made with absolute certainty from these data. Nevertheless, the correlations found are surprisingly good, given that the studies included span over 30 years, with possible differences in donor characteristics, cell culture techniques, growth media, explanation methods and selection of biopsy areas.

Growth media are also well-recognized variables affecting cell growth and proliferation. Varying concentrations of serum used in culture result in different growth effects, as demonstrated by Holley et al.40, with the number of PDs achieved directly related to the original concentration of serum added. Studies varied in terms of serum type and concentration used; for example, EC were cultured with 30% human serum in the study by Watkins et al.41 while 15% fetal bovine serum was used in the study by Fukai et al.42.

Studies also varied in terms of their definition of senescence, ranging from: senescence-associated morphological changes, 50% positive for SA-beta-galactosidase staining, or less than one PD occurring within three weeks after subculture. Diverse interpretations of ceased proliferation may have led to inconsistencies of the recorded replicative capacity.

Vessel type is an additional variable known to affect cellular replicative ability; for example, telomere length has been reported to decrease more rapidly in arterial rather than venous endothelial cells 15. It has even been shown that the segment of the vessel used affects telomere attrition; for example, the distal versus the proximal segment of the abdominal aorta showing accelerated telomere attrition 16. This most likely reflects the hemodynamic stress factor 43444546.

The scatter plots showing the effect of vessel type and gender showed the data-points to be generally equally distributed. Any exceptions were most likely due to factors associated with disease, shear stress or age, as different studies varied in terms of sample characteristics.

It would be misleading to perform a Funnel test (to test for publication bias) or a Cochrane-Q test (to test for heterogeneity between studies included). Different researchers focused on specific age ranges or disease states; therefore, any asymmetry observed in a funnel plot will probably not be due to systematic error or publication bias, but be due to factors associated with disease, age or sheer stress.

It should also be considered that studies included in this analysis did not always specify the site from which the cells were explanted. Moreover, the removal of vascular tissue is much less likely to occur in healthy individuals, compared to fibroblasts (which are far easier to obtain), unless the donor had died in an accident. It is, therefore, uncertain that all of the donors classed as ‘without cardiovascular-related disease’ included in this study were entirely healthy. For example, it is possible that senescence in endothelial or vascular smooth muscle cells is also dependent on damage induced by other, unrelated pathological conditions in the individual.

Despite all of this, the plots still show a clear inverse relationship between donor age and proliferative capacity in vitro. These conclusions are supported by the T-test values and correlation coefficients.

Conclusions

This survey of the available literature demonstrates that a clear inverse relationship exists between the replicative capacity of vascular endothelial and smooth muscle cells and donor age. Individuals free of cardiovascular-related diseases show a greater replicative capacity at given ages, than those with defined pathologies. The mean CPD for each diseased age group roughly corresponds to that of older, non-diseased subjects. This suggests that disease state rather than donor age is the primary variable correlating with senescence. Perhaps surprisingly, individuals with cardiovascular disease appear to start off with a lower cellular replicative capacity in early life, than non-diseased individuals. The difference in average CPDs in age-matched individuals in the two groups is roughly constant throughout the lifetime. This is consistent with the hypothesis that individuals differ in terms of inherited cellular replicative capacity, but exhaust their proliferative potential at similar rates.

Abbreviations

CPD: Cumulative Population Doublings; DNA: Deoxyribonucleic acid; EC: Endothelial Cell; HUVEC: Human Umbilical Vein Endothelial Cells; IL: 1α-Interleukin-1 alpha; ICAM-1: Intracellular Adhesion Molecule 1; NA: Not Applicable; PAI-1: Plasminogen Activator Inhibitor-1; TERT: Telomerase Reverse Transcriptase; VSMC: Vascular Smooth Muscle Cell.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

RF devised, planned and assigned this project to MK to satisfy the criteria of completing a BSc. MK collected and analyzed the data and drafted the manuscript under the supervision of RF. All authors read and approved the final manuscript.

<p>Cyclin-dependent kinase inhibitor p16(INK4a) and telomerase may co-modulate endothelial progenitor cells senescence</p> Yang DG Liu L Zheng XY Ageing Res Rev 2008 7 137 146 10.1016/j.arr.2008.02.001 18343732 <p>Vascular aging: insights from studies on cellular senescence, stem cell aging, and progeroid syndromes</p> Minamino T Komuro I Nat Clin Pract Cardiovasc Med 2008 5 637 648 10.1038/ncpcardio1324 18762784 <p>Vascular smooth muscle cells undergo telomere-based senescence in human atherosclerosis: effects of telomerase and oxidative stress</p> Matthews C Gorenne I Scott S Figg N Kirkpatrick P Ritchie A Goddard M Bennett M Circ Res 2006 99 156 164 10.1161/01.RES.0000233315.38086.bc 16794190 <p>Microarray analysis of senescent vascular smooth muscle cells: a link to atherosclerosis and vascular calcification</p> Burton DG Giles PJ Sheerin AN Smith SK Lawton JJ Ostler EL Rhys-Williams W Kipling D Faragher RG Exp Gerontol 2009 44 659 665 10.1016/j.exger.2009.07.004 19631729 <p>Clearance of p16Ink4a-positive senescent cells delays ageing-associated disorders</p> Baker DJ Wijshake T Tchkonia T LeBrasseur NK Childs BG van de Sluis B Kirkland JL van Deursen JM Nature 2011 479 232 236 10.1038/nature10600 3468323 22048312 <p>Replicative lifespan of cultivated human cells. Effects of donor's age, tissue and genotype</p> Martin GM Sprague CA Epstein CJ Lab Invest 1970 23 86 92 5431223 <p>The relationship between <it>in vitro</it> cellular aging and <it>in vivo</it> human age</p> Schneider EL Mitsui Y Proc Natl Acad Sci U S A 1976 73 3584 3588 10.1073/pnas.73.10.3584 431162 1068470 <p>Chronologic and physiologic age affect replicative life-span of fibroblasts from diabetic, prediabetic, and normal donors</p> Goldstein S Moerman EJ Soeldner JS Gleason RE Barnett DM Science 1978 199 781 782 10.1126/science.622567 622567 <p>Relationship between donor age and the replicative lifespan of human cells in culture: a reevaluation</p> Cristofalo VJ Allen RG Pignolo RJ Martin BG Beck JC Proc Natl Acad Sci U S A 1998 95 10614 10619 10.1073/pnas.95.18.10614 27943 9724752 <p>Relation between replicative senescence of human fibroblasts and life history characteristics</p> Maier AB Westendorp RG Ageing Res Rev 2009 8 237 243 10.1016/j.arr.2009.01.004 19491042 <p>Persistence of high-replicative capacity in cultured fibroblasts from nonagerians</p> Maier AB le Cessie S de Koning-Treurniet C Blom J Westendorp RG van Heemst D Aging Cell 2007 6 27 33 10.1111/j.1474-9726.2006.00263.x 17266674 <p>A biomarker that identifies senescent human cells in culture and in aging skin <it>in vivo</it></p> Dimri GP Lee X Basile G Acosta M Scott G Roskelley C Medrano EE Linskens M Rubelj I Pereira-Smith O Proc Natl Acad Sci U S A 1995 92 9363 9367 10.1073/pnas.92.20.9363 40985 7568133 <p>Cellular senescence in aging primates</p> Herbig U Ferreira M Condel L Carey D Sedivy JM Science 2006 311 1257 10.1126/science.1122446 16456035 <p>Long-term caloric restriction delays age-related decline in proliferation capacity of murine lens epithelial cells <it>in vitro</it> and <it>in vivo</it></p> Li Y Yan Q Wolf NS Invest Ophthalmol Vis Sci 1997 38 100 107 9008635 <p>Cellular senescence after single and repeated balloon catheter denudations of rabbit carotid arteries</p> Fenton M Barker S Kurz DJ Erusalimsky JD Arterioscler Thromb Vasc Biol 2001 21 220 226 10.1161/01.ATV.21.2.220 11156856 <p>Age dependent aneuploidy and telomere length of the human vascular endothelium</p> Aviv H Khan MY Skurnick J Okuda K Kimura M Gardner J Priolo L Aviv A Atherosclerosis 2001 159 281 287 10.1016/S0021-9150(01)00506-8 11730807 <p>Apoptosis of human vascular smooth muscle cells derived from normal vessels and coronary atherosclerotic plaques</p> Bennet MR Evan GI Schwartz SM J Clin Invest 1995 95 2266 2274 10.1172/JCI117917 295839 7738191 <p>Synthesis of IL-1 alpha and IL-1 beta by arterial cells in atherosclerosis</p> Moyer CF Sajuthi D Tulli H Williams JK Am J Pathol 1991 138 951 960 1886091 2012178 <p>Increased type 1 plasminogen activator inhibitor gene expression in atherosclerotic human arteries</p> Schneiderman J Sawdey MS Keeton MR Bordin GM Bernstein EF Dilley RB Loskutoff DJ Proc Natl Acad Sci U S A 1992 89 6998 7002 10.1073/pnas.89.15.6998 49632 1495992 <p>Adhesion molecules on the endothelium and mononuclear cells in human atherosclerotic lesions</p> van der Wal AC Das PK Tigges AJ Becker AE Am J Pathol 1992 141 1427 1433 1886778 1281621 <p>Pathophysiology of vascular calcification: Pivotal role of cellular senescence in vascular smooth muscle cells</p> Burton DG Matsubara H Ikeda K Exp Gerontol 2010 45 819 824 10.1016/j.exger.2010.07.005 20647039 <p>Telomere length and replicative aging in human vascular tissues</p> Chang E Harley CB Proc Natl Acad Sci U S A 1995 92 11190 11194 10.1073/pnas.92.24.11190 40597 7479963 <p>Genetic determination of telomere size in humans: a twin study of three age groups</p> Slagboom PE Droog S Boomsma DI Am J Hum Genet 1994 55 876 882 1918314 7977349 <p>Telomere length, risk of coronary heart disease, and statin treatment in the West of Scotland Primary Prevention Study: a nested case–control study</p> Brouilette SW Moore JS McMahon AD Thompson JR Ford I Shepherd J Packard CJ Samani NJ West of Scotland Coronary Prevention Study Group Lancet 2007 369 107 114 10.1016/S0140-6736(07)60071-3 17223473 <p>Association between telomere length in blood and mortality in people aged 60 years or older</p> Cawthon R Smith KR O'Brien E Sivatchenko A Kerber RA Lancet 2003 361 393 395 10.1016/S0140-6736(03)12384-7 12573379 <p>The signals and pathways activating cellular senescence</p> Ben-Porath I Weinberg RA Int J Biochem Cell Biol 2005 37 961 976 10.1016/j.biocel.2004.10.013 15743671 <p>Cellular and molecular mechanisms of stress-induced premature senescence (SIPS) of human diploid fibroblasts and melanocytes</p> Toussaint O Medrano EE von Zglinicki T Exp Gerontol 2000 35 927 945 10.1016/S0531-5565(00)00180-7 11121681 <p>Substantial variation in qPCR measured mean blood telomere lengths in young men from eleven European countries</p> Eisenberg DT Salpea KD Kuzawa CW Hayes MG Humphries SE European Atherosclerosis Research Study II Group Am J Hum Biol 2011 23 228 231 10.1002/ajhb.21126 21319253 <p>Leukocyte telomeres are longer in African Americans than in whites: the National Heart, Lung, and Blood Institute Family Heart Study and the Bogalusa Heart Study</p> Hunt SC Chen W Gardner JP Kimura M Srinivasan SR Eckfeldt JH Berenson GS Aviv A Aging Cell 2008 7 451 458 10.1111/j.1474-9726.2008.00397.x 2810865,2810865 18462274 <p>Telomeres and telomerase in the fetal origins of cardiovascular disease: a review</p> Demerath E Cameron N Gillman MW Towne B Siervogel RM Hum Biol 2004 76 127 146 10.1353/hub.2004.0018 2801408 15222684 <p>Accelerated telomere shortening in response to life stress</p> Epel ES Blackburn EH Lin J Dhabhar FS Adler NE Morrow JD Cawthon RM Proc Natl Acad Sci U S A 2004 101 17312 17315 10.1073/pnas.0407162101 534658 15574496 <p>Offspring’s Leukocyte telomere length, paternal age, and telomere elongation in sperm</p> Kimura M Cherkas LF Kato BS Demissie S Hjelmborg JB Brimacombe M Cupples A Hunkin JL Gardner JP Lu X Cao X Sastrasinh M Province MA Hunt SC Christensen K Levy D Spector TD Aviv A PLoS Genet 2008 4 e37 10.1371/journal.pgen.0040037 2242810 18282113 <p>The limited <it>in vitro</it> lifetime of human diploid cell strains</p> Hayflick L Exp Cell Res 1965 37 614 636 10.1016/0014-4827(65)90211-9 14315085 Arking R The Biology of Aging: Observations and Principles Sunderland, MA, USA: Sinauer Associates, Inc 2 1998 <p>Replicative senescence of vascular smooth muscle cells enhances the calcification through initiating the osteoblastic transition</p> Nakano-Kurimoto R Ikeda K Uraoka M Nakagawa Y Yutaka K Koide M Takahashi T Matoba S Yamada H Okigaki M Matsubara H Am J Physiol Heart Circ Physiol 2009 297 H1673 1684 10.1152/ajpheart.00455.2009 19749165 <p>Increased expression of extracellular proteins as a hallmark of human endothelial cell <it>in vitro</it> senescence</p> Hampel B Fortschegger K Ressler S Chang MW Unterluggauer H Breitwieser A Sommergruber W Fitzky B Lepperdinger G Jansen-Dürr P Voglauer R Grillari J Exp Gerontol 2006 41 474 481 10.1016/j.exger.2006.03.001 16626901 <p>Cellular senescence in endothelial cells from atherosclerotic patients is acceleration by oxidative stress associated with cardiovascular risk factors</p> Vogel G Thorin-Trescases N Farhat N Nguyen A Villeneuve L Mamarbachi AM Fortier A Perrault LP Carrier M Thorin E Mech Ageing Dev 2007 128 662 671 10.1016/j.mad.2007.09.006 18022214 <p>Telomere attrition and accumulation of senescent cells in cultured human endothelial cells</p> Hastings R Qureshi M Verma R Lacy PS Williams B Cell Prolif 2004 37 317 324 10.1111/j.1365-2184.2004.00315.x 15245567 <p>Efficient expression of exogenous genes in primary vascular cells using IRES-based retroviral vectors</p> Garton KJ Ferri N Raines EW Biotechniques 2002 32 830 834 11962605 <p>Control of the initiation of DNA synthesis in 3T3 cells: Low-molecular-weight nutrients</p> Holley RW Kiernan JA Proc Nat Acad Sci U S A 1974 71 2942 2945 10.1073/pnas.71.8.2942 <p>Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for Use in endothelial seeding of vascular prostheses</p> Watkins MT Sharefkin JB Zajtchuk R Maciag TM D'Amore PA Ryan US Van Wart H Rich NM J Surg Res 1983 36 588 596 <p>Human arterial smooth muscle cell strains derived from patients with moyamoya disease: changes in biological characteristics and proliferative response during cellular ageing <it>in vitro</it></p> Fukai N Aoyagi M Yamamoto M Sakamoto H Ogami K Matsushima Y Yamamoto K Mech Ageing Dev 1994 75 21 33 10.1016/0047-6374(94)90025-6 9128751 <p>Does high shear stress induced by blood flow lead to atherosclerosis?</p> Roach MR Smith NB Perspect Biol Med 1983 26 287 303 6341962 <p>Fluid dynamics as a factor in the localization of atherogenesis</p> Nerem RM Levesque MJ Ann N Y Acad Sci 1983 416 709 710 10.1111/j.1749-6632.1983.tb35222.x 6587822 <p>Hemodynamic shear stress and the endothelium in cardiovascular pathophysiology</p> Davies PF Nat Clin Pract Cardiovasc Med 2009 6 16 26 10.1038/ncpcardio1397 2851404 19029993 <p>Telomere attrition of the human abdominal aorta: relationships with age and atherosclerosis</p> Okuda K Khan MY Skurnick J Kimura M Aviv H Aviv A Atherosclerosis 2000 152 391 398 10.1016/S0021-9150(99)00482-7 10998467 <p>Human smooth muscle cells cultured from atherosclerotic plaques and uninvolved vessel wall</p> Eskin SG Sybers HD Lester JW Navarro LT Gotto AM DeBakey ME In Vitro 1981 17 713 718 10.1007/BF02628408 7327599 <p>Comparative endocrinology-paracrinology-autocrinology of human adult large vessel endothelial and smooth muscle cells</p> Hoshi H Kan M Chen J McKeehan W In Vitro Cell Dev Biol 1987 24 309 320 <p>The effect of donor Age on the <it>in vitro</it> life span of cultured human arterial smooth-muscle cells</p> Bierman EL In Vitro 1978 14 951 955 10.1007/BF02616126 730203 <p>Isolation, growth requirements, cloning, prostacyclin production and life-span of human adult endothelial cells in low serum culture medium</p> Hoshi H McKeehan WL In Vitro Cell Dev Biol 1986 22 51 56 10.1007/BF02623441 3080403 <p>Cultured endothelial cells derived from the human iliac arteries</p> Glassberg MK Bern MM Coughlin SR Haudenschild CC Hoyer L Antoniades HN Zetter BR In Vitro 1982 18 859 866 10.1007/BF02796327 6816718 <p>Karyotypic and phenotypic changes during <it>in vitro</it> aging of human endothelial cells</p> Johnson TE Umbenhauer DR Hill R Bradt C Mueller SN Levine EM Nichols WW J Cell Physiol 1992 150 17 27 10.1002/jcp.1041500104 1309825 <p>Serial propagation of human endothelial cells <it>in vitro</it></p> Maciag T Hoover GA Stemerman MB Weinstein R J Cell Biol 1981 91 420 426 10.1083/jcb.91.2.420 2111986 7309790 <p>Evaluation of long-term cultured endothelial cells as a model system for studying vascular ageing</p> Nobuhiko H Yamamoto M Imamura T Mitsui Y Yamamoto K Mech Ageing Dev 1988 46 111 123 10.1016/0047-6374(88)90119-4 3067000 <p>Chronic treatment with N-acetyl-cystein delays cellular senescence in endothelial cells isolated from a subgroup of atherosclerotic patients</p> Vogel G Thorin-Trescases N Farhat N Mamarbachi AM Villeneuve L Fortier A Perrault LP Carrier M Thorin E Mech Ageing Dev 2008 129 261 270 10.1016/j.mad.2008.01.004 18302967