Early accelerated senescence of circulating endothelial progenitor cells in premature coronary artery disease patients in a developing country - a case control study
BMC Cardiovascular Disorders
Early accelerated senescence of circulating endothelial progenitor cells in premature coronary artery disease patients in a developing country - a case control study
Kranthi Vemparala 0
Ambuj Roy 2
Vinay Kumar Bahl 2
Dorairaj Prabhakaran 1
Neera Nath 6
Subrata Sinha 5
Pradipta Nandi 2
Ravindra Mohan Pandey 4
Kolli Srinath Reddy 3
Ajay Manhapra 7
Ramakrishnan Lakshmy 0
0 Department of Cardiac Biochemistry, All India Institute of Medical Sciences , New Delhi , India
1 Center for Chronic Disease Control and CARRS COE, Public Health Foundation of India , New Delhi , India
2 Department of Cardiology, All India Institute of Medical Sciences , New Delhi , India
3 Public Health Foundation of India , New Delhi , India
4 Department of Biostatistics, All India Institute of Medical Sciences , New Delhi , India
5 National Brain Research Center , Manesar, Haryana , India
6 Department of Biochemistry, All India Institute of Medical Sciences , New Delhi , India
7 Department of Medicine, University of Virginia School of Medicine , Charlottesville, VA , USA
Background: The decreased number and senescence of circulating endothelial progenitor cells (EPCs) are considered markers of vascular senescence associated with aging, atherosclerosis, and coronary artery disease (CAD) in elderly. In this study, we explore the role of vascular senescence in premature CAD (PCAD) in a developing country by comparing the numerical status and senescence of circulating EPCs in PCAD patients to controls. Methods: EPCs were measured by flow cytometry in 57 patients with angiographically documented CAD, and 57 controls without evidence of CAD, recruited from random patients 50 years of age at All India Institute of Medical Sciences. EPC senescence as determined by telomere length (EPC-TL) and telomerase activity (EPC-TA) was studied by real time polymerase chain reaction (q PCR) and PCR- ELISA respectively. Result: The number of EPCs (0.18% Vs. 0.039% of total WBCs, p < 0.0001), and EPC-TL (3.83 Vs. 5.10 kb/genome, p = 0.009) were markedly lower in PCAD patients compared to controls. These differences persisted after adjustment for age, sex, BMI, smoking and medications. EPC-TA was reduced in PCAD patients, but was statistically significant only after adjustment for confounding factors (1.81 Vs. 2.20 IU/cell, unadjusted p = 0.057, adjusted p = 0.044). Conclusions: We observed an association between increased vascular cell senescence with PCAD in a sample of young patients from India. This suggests that early accelerated vascular cell senescence may play an important mechanistic role in CAD epidemic in developing countries like India where PCAD burden is markedly higher compared to developed countries.
Premature coronary artery disease; Endothelial progenitor cells; Senescence; Telomere length; Telomerase activity
While coronary vascular disease (CVD) and coronary
artery disease (CAD) occurs predominantly among older
individuals in developed countries, it occurs at earlier
ages in developing countries. The proportion of CVD
deaths below 70 years of age in 1990 was only 22.8% in
the established market economies, whereas it was 52.2%
in India . It is unclear why communities where CAD
is emerging as a threat have a high burden of premature
coronary artery diseases (PCAD) .
The development of CAD among elderly has been
associated with the progression of senescence in almost all
cellular elements of the vascular system [3,4].
Endothelial dysfunction is presumed to play a crucial role in the
development of atherosclerosis and coronary artery
disease spectrum including PCAD . Circulating
endothelial progenitor cells (EPCs) mobilized from the bone
marrow by the vascular system provide an endogenous
repair mechanism for endothelial cell injury , and may
therefore have an important role in the pathophysiology
of CAD progression. Numerical decline and functional
impairment of circulating EPCs has been associated with
aging and senescence of vascular system, and more
pronounced numerical and functional decline of EPCs have
been associated with CAD among elderly [3,7-9]. Reduced
telomere length is considered a marker of senescence in
various cells including those in vascular system. Reduced
telomere length in EPCs (EPC-TL) is also associated with
CAD among elderly [3,7-9]. In addition, increased EPC
senescence has been shown to be associated with higher
risk for cardiovascular events in individuals , and risk
factors and clinical conditions like diabetes and
hypertension are known to have detrimental effect on numerical
and functional levels of EPCs in CAD patients [11-15].
Based on demonstration of extensive association of
vascular cell senescence with atherosclerosis and CAD, and
evidence of shared biochemical pathways between vascular
aging and atherosclerosis, others have suggested that
CAD among elderly can be viewed as accelerated aging
[4,16]. However, the role of accelerated vascular aging in
the pathogenesis of PCAD has not been explored before
in detail especially in the setting of a developing country
like India where the burden of PCAD is very high. In this
study we explore the role of numerical and functional
decline of EPCs, and telomere shortening of EPCs, markers
of vascular senescence in PCAD patients from India.
The Ethics Committee of the All India Institute of
Medical Sciences (New Delhi, India), approved the protocol
and written informed consent was obtained from all
subjects prior to the study. A total of 57 subjects, age
50 years of either sex, with evidence of CAD as
confirmed by the presence of either > 50% stenosis in the left
main coronary artery or > 70% stenosis in other arteries
on coronary angiography were recruited for the study
group. Fifty-seven age matched ( 5 years) non-diabetic
subjects of both genders without known heart disease
and either having a normal coronary angiogram or
without demonstrable ischemia on stress testing were
recruited as controls from random patients visiting
Cardiology department of All India Institute of Medical
Sciences (AIIMS), New Delhi, India from 2008 to 2010.
Subjects with any form of cancer, renal disease,
rheumatoid arthritis, pneumonia, nephropathy and
cerebrovascular disease were excluded. 30 ml fasting venous blood
sample was collected in EDTA tubes. 200 l of the whole
blood was used for EPC enumeration. Plasma was
separated from 3 ml of blood for biochemical analysis. Rest
of the blood was used for EPC isolation for
measurement of telomere length and telomerase activity. Insulin,
hsCRP (high sensitive C-reactive protein), and
homocysteine were analyzed by ELISA using kits from
Mercodia (Sweden), Biochek (CA) and Diazyme (CA)
respectively. Glucose was measured by glucose oxidase,
total cholesterol by CHOD-PAP, and triglycerides by
GPO-PAP methods using kits from RANDOX (Crumlin,
UK). High density lipoprotein (HDL) cholesterol was
measured by precipitation method using RANDOX kits.
Insulin resistance was calculated using Homeostatic
Model assessment (HOMA). Low Density Lipoprotein
(LDL) was calculated using friedwalds formula.
EPCs were quantified in blood by flow cytometry. Briefly
200 l of peripheral blood was treated with FcR blocking
reagent (MiltenyiBiotec, Germany) and incubated with
Fluorescein isothiocyanate (FITC) labeled CD34 (BD
Biosciences, USA) and R-phycoerythrin (PE) labeled VEGFR2
antibodies (R&D systems, USA). Labeled cells were lysed
with Fluorescence-activated cell sorting (FACS) lysis
buffer (BD Biosciences, USA) and fixed. Baseline parameters,
compensation settings and gates were set using unstained
controls and isotype controls. EPCs were identified as
CD34+/KDR + dual positive cells in the upper right
quadrant region. A total of 500,000 events were recorded in
each unstained and stained sample. The numbers of
CD34+/KDR + cells were expressed as a percentage of
Mononuclear cells (MNC) were isolated from blood
samples by HISTOPAQUE-1077 (Sigma chemicals,
India) density gradient centrifugation according to
manufacturers protocol. EPCs were isolated using a two step
protocol of Magnetic Activated Cell Sorting (MACS)
using CD 34 multisort kit (MiltenyiBiotec, Germany)
and indirect labeling procedure with PE labeled VEGFR2
(R&D systems, India) and anti-PE micro beads (Miltenyi
Biotec, Germany). Briefly, MNC were treated with FcR
blocking reagent (Miltenyi Biotec, Germany) and
incubated with CD34 multisort beads and PE labeled VEGFR2.
The CD34 labeled cells were separated through positive
selection and incubated with anti-PE microbeads and cells
which were dual positive for CD34/VEGFR2 collected,
counted and aliquoted for telomere length and telomerase
Measurement of EPC Telomere length (EPC-TL)
EPC-TL was measured by real time PCR method as
described by Cawthon et al.  Callaghan et al.  and
N Callaghan and Fenech  with minor modifications.
Measurement of EPC Telomerase activity (EPC-TA)
EPC Telomerase activity (EPC-TA) was measured by a
combination of telomeric repeat amplification protocol
(TRAP) and photometric enzyme immunoassay according
to manufacturers protocol (Roche Diagnostics, India).
Statistical analysis was performed using STATA
statistical software (version 9.0 for windows). Fifty patients
and 50 controls were needed for 90% power and of
0.05. We elected to recruit additional subjects within a
stipulated time and a ceiling of 60. Normality of the
sampling distribution of each variable was tested using
Kolmogorov-Smirnov test for normality. The
distributions of EPC number, EPC-TL, and EPC-TA were not
normal and therefore log transformed for analysis.
Continuous variables like age, height, weight, BMI, systolic
blood pressure, diastolic blood pressure, medication
dose, left ventricular ejection fraction (LVEF)% and
biochemical estimates were expressed as Mean SD
(Standard Deviation) and categorical variables were expressed
as numbers and percentage. Log transformed variables
were expressed as geometric mean with 95% confidence
intervals (95% CI). Baseline characteristics and
biochemical estimates of cases and controls were compared
using unpaired students t test. Categorical variables that
were not normally distributed were analyzed using
Wilcoxon rank sum test. Students t test was used to
compare the means of EPC number, EPC-TL, and
EPCTA in cases and controls. Linear regression analysis was
employed to adjust for confounding variables. Bivariate
and partial correlations were computed for assessing
correlations between EPC number/EPC senescence and
biochemical parameters. Confounding variables taken for
adjustment included age, sex, BMI, smoking and
medications. Statistical significance was assumed if P value was
less than or equal to 0.05.
The baseline characteristics are detailed in Tables 1 and 2.
The proportion of female patients were low in both PCAD
(1) or control group (4), and family history of CAD was
more often present in PCAD group. Use of statins,
ACEinhibitors, blockers and aspirin was significantly higher
in PCAD. PCAD group had lower mean total cholesterol,
LDL, HDL and triglycerides, possibly reflecting higher
statin use, but had significantly higher homocysteine levels
compared to controls.
Circulating EPC number and senescence
Figure 1A and 1B shows the flow cytometry analysis of
circulating EPCs. Figure 1B shows the stained EPCs. As
shown in Table 3 the mean percent of EPCs were
significantly lower in PCAD patients compared to controls,
and this persisted after adjustment for confounding
variables. The mean EPC-TL was also markedly lower in
PCAD patients compared to controls and the difference
Table 1 Baseline characteristics of Subjects
Family history for CAD (n) (%)
Family history for
hypertension (n) (%N)
ACE Inhibitors, n (%)
blockers, n (%)
LVEF% (mean SD)
Hypertension, n (%)
127.9 11.88 127.46 14.87
remained significant after adjustment. The mean relative
EPC-TA was lower in PCAD patients as compared to
controls, but the difference was statistically significant
(P = 0.044) only after adjusting for confounding
variables. Additionally adjusting for family history of
diabetes did not change these associations. The EPC
numbers were lower in smokers as compared to non
smokers (0.022% vs. 0.014%) but the difference was not
statistically significant (p = 0.127).
Table 2 Biochemical characteristics of subjects
Total cholesterol (mg/dl)
Figure 1 Unstained (A) and stained (B) quadrangle plot of CD34-Fluorescence isothiocyanate in forward scatter and versus VEGRF2
(KDR)-phycoerythrin in the side scatter in one representative patient sample. The upper right quadrant shows the dual stained EPCs.
Correlation of biochemical parameters with circulating
EPC levels and EPC senescence
In controls, EPC number positively correlated with total
cholesterol before and after adjusting for age, sex, BMI
and smoking (Unadjusted Pearson r = 0.231, P = 0.021,
Adjusted Pearson r = 0.218, P = 0.033). However the
association was lost when adjusted for medications. HDL
levels were positively correlated with EPC number
(Unadjusted Pearson r = 0.284, P = 0.004, adjusted r = 0.241,
P = 0.018). In PCAD patients EPC numbers negatively
correlated with triglyceride levels (unadjusted Pearson
r = 0.280, p = 0.049 and adjusted Pearson r = 0.380,
Table 3 Number and senescence of circulating endothelial progenitor cells in premature CAD patients compared to
Adjusted P value*
EPC telomere length (kb/genome)
EPC telomerase activity (IU/cell)
*Adjusted for age, sex, BMI, smoking and medication.
P = 0.010). EPC-TL was also correlated with triglycerides
(Unadjusted Pearson r = 0.326, P = 0.014 and adjusted
r = 0.289, P = 0.038). EPC-TL positively correlated with
EPC-TA (Pearson r = 0.409, P = 0.002).
We observed markedly lower numbers of circulating
EPCs in young patients with angiographically
documented CAD from India as compared to controls
without CAD. In addition, the EPCs from PCAD patients
also showed a marked decrease in telomere length and
telomerase activity. The difference remained even after
adjusting for all the confounding variables (age, sex,
BMI, use of medication and smoking). Our findings
suggest that PCAD patients in India have accelerated
vascular senescence compared to those without CAD. EPC
number and telomere length also negatively correlated
with serum triglyceride levels in patients.
The results reported in the present study are in
agreement with other studies reporting a reduced EPC
number in older CAD patients. Vasa et al.  reported
lower EPC (CD34/KDR positive) numbers and function
in CAD patients with a mean age of 62 years. Eizawa
et al. has also reported a similar reduction in EPC
(CD34+ cells) number in CAD patients . Aging
associated numerical and functional decline in EPCs in CAD
patients has been attributed to exhaustion of
stem/progenitor cells in the bone marrow due to chronic vascular
injury, reduced mobilization, diminished migratory and
adhesion capacity of EPCs and deregulation of EPC
differentiation [22,23], and is thought to render the elderly
more prone to endothelial dysfunction and
cardiovascular disease . Results of our study suggest that low
EPC levels may be responsible for endothelial
dysfunction and atherosclerosis in PCAD patients as well.
A shorter EPC-TL in PCAD patients reported in the
present study points towards accelerated senescence of
EPCs resulting in lower EPC number, suggestive of
compromised repair of dysfunctional endothelium. EPC
telomere shortening and decreased EPC telomerase activity
has been reported in older patients with stable and
unstable CAD [25,26]. Kushner et al. reported age related
decline of EPC-TL in healthy men from a developed
nation, starting after the age of 55 years, and EPC-TL was
20% shorter in older as compared to middle aged men
and young suggesting EPC telomere shortening may be
responsible for age related endothelial dysfunction .
In our study, the PCAD patients with a mean age of
43 years show a similar decline in TL (25% reduction)
compared to controls of similar age, suggesting an
earlier onset of EPC senescence in them. Increased oxidative
stress associated with metabolic derangements has been
suggested as an explanation for the shorter EPC
telomeres and EPC lower telomerase activity , and this
could be applicable to younger patients too. We did not
measure markers of oxidative stress in our study. The
negative correlation between triglycerides and EPC
number and senescence on our study would be relevant as
Indians are known to consume high carbohydrate diet,
and have higher triglyceride levels. The PCAD patients
showed lower triglyceride levels possibly due to statin
use . Other authors have also reported an association
between EPC and triglycerides . Triglyceride rich
lipoproteins have been shown to increase senescence of
endothelial progenitor cells via oxidative stress .
Since the subjects in this study had a mean age of 43,
age is unlikely to be the culprit for low EPC count and
higher level of EPC senescence in PCAD patients. This
EPC senescence is probably driven by multiple factors
including increased oxidative stress due to smoking, and
atherogenic lipoprotein phenotype characterized by high
triglycerides and low HDL.
Recent studies have shown that diet may have
significant influence on EPC-TL and EPC-TA . As with
cardiac risk factors, non-pharmacologic intervention like
exercise [30,31], and diet modification [29,32] can
positively influence vascular aging along with medications
like statins . Statins are known to promote EPC
mobilization, proliferation, migration, adhesion, and
differentiation, and reduce senescence. We did not find any
significant correlation between dose of statins and EPC
number and EPC senescence in our study possibly
because most of our subjects were on low dose of statins.
Though prematurely speculative, this study with the
context of prior evidence from developed countries
provides interesting insights into the mechanisms of
premature CVD in Indians. Autopsy studies from United
States have shown that coronary atherosclerosis have
markedly declined from 77% prevalence in 1950s to
8.5% in 2011, a period of sharp decline in premature
CAD death and risk factor prevalence in United States
, suggesting that the high CAD burden among
middle aged adults in 1940s and 1950s in United States (as
seen now in countries like India and China) was
probably driven by reservoir of atherosclerosis in the young
population, and the abundance of noxious risk factors
. Aging associated atherosclerosis can be considered
as accelerated vascular aging probably influenced by
additional pathological burden induced by various other
noxious environments like smoking, dyslipidemia, and
hypertension , and atherosclerosis occurring at
younger age could be due to early accelerated aging of
the vascular wall . Consistent with this view, our data
suggests that senescence of vascular system may be
happening early in an accelerated fashion in PCAD patients
from developing countries. Nilsson et al. has suggested
that the early vascular aging including telomere
anomalies that lead to atherosclerosis is probably a reflection of
more generalized early biological aging in susceptible
individuals, and this susceptibility is partly driven stresses
of early life like fetal health and early childhood adverse
growth patterns , a situation still common in
countries like India. Putting all of this together, it is tempting
to speculate that as survival rates at young ages in
developing countries increase and the high exposure to fetal
and early life stresses persist, the reservoir of
susceptible people with early vascular aging (and general
biological aging) in developing country like India can be
very high. The exponentially increasing prevalence of
atherogenic risk factors at young adulthood in developing
countries  may be further accelerating the vascular
senescence in the population, especially among the already
susceptible ones. This complex interplay between
earlyaccelerated vascular senescence biology and the
environmental factors like urbanization, nutrition, and ecology of
living environments is probably a crucial driver of the
epidemic of premature CAD in developing countries.
We have not excluded patients who are on statin
therapy, which influence EPC number. We have however
adjusted for the effect of statins and other medications in
statistical analysis. Further, we have not looked at the
functional and mobilization capacity of EPCs in
premature CAD patients, possibly of greater importance than
absolute EPC number and needs to be addressed in
future studies. Unlike many prior studies EPC-TL and
EPC-TA in our study was measured directly in EPC
isolated from individuals, and not from EPC cultures, and
this more realistically mimic in-vivo conditions.
Moreover the specific role of EPCs in development of
atherosclerosis and CAD is still being clarified.
Significantly lower EPCs in premature CAD patients in
the present study suggest impaired repair mechanism
predisposing to endothelial dysfunction at younger ages.
Association of EPC senescence with triglycerides, which
is part of the atherogenic South Asian phenotype, is
suggestive of a possible role of classical risk factors in
regulating EPC number in this population. A shorter EPC
telomere length and a reduced telomerase activity, in the
young CAD patients in the present study, points to an
accelerated senescence of EPCs in vivo, resulting in
lower circulating EPCs. Together with prior data, our
study suggests that early-accelerated vascular senescence
may be a crucial driver of premature CAD in developing
countries. The study also suggests that EPC telomere
length could be used as an early marker for detecting
impaired repair mechanism predisposing to endothelial
dysfunction in premature CAD patients. The pliability of
vascular aging to control of risk factors, and the possible
involvement of early life factors in the generation of
early accelerated vascular senescence/aging suggests that
national policies focusing simultaneously on early life
enhancement and chronic risk factor reduction maybe
likely to curtail the burden of premature CAD in
BMI: Body mass index; CVD: Coronary vascular disease; CAD: Coronary artery
disease; EPCs: Endothelial progenitor cells; EPC-TL: Endothelial progenitor cell
telomere length; EPC-TA: Endothelial progenitor cell telomerase activity;
EDTA: Ethylene diamine tetra acetic acid; FACS: Fluorescence-activated cell
sorting; FITC: Fluorescein isothiocyanate; hsCRP: High sensitive C-reactive
protein; HDLc: High density lipoprotein cholesterol; HOMA: Homeostatic
Model assessment; LDLc: Low density lipoprotein cholesterol; LVEF: Left
ventricular ejection fraction; MACS: Magnetic activated cell sorting;
MNC: Mononuclear cells; PCAD: Premature coronary artery disease;
PCRELISA: Polymerase chain reaction enzyme linked immunoabsorbent assay;
PE: R-phycoerythrin; qPCR: Real time polymerase chain reaction;
TRAP: Telomeric repeat amplification protocol; WBCs: White blood cells.
KV carried out the experiments, performed the statistical analysis and drafted
the manuscript. LR conceived the study, participated in its design and
coordination and helped to draft the manuscript. DP helped in designing
the study and gave intellectual inputs for writing the manuscript. VKB, PN
and AR helped in recruitment of subjects. SS and NN helped in experimental
design. RMP helped in performing the statistical analysis. AM gave
intellectual inputs and edited the manuscript. KSR provided intellectual
inputs for study design. All authors read and approved the final manuscript.
1. Murray CJL , Lopez AD : Global comparative assessments in the health sector: disease burden, expenditures, and intervention packages: collected reprints from the Bulletin of the World Health Organization . Geneva: World Health Organization ; 1994 .
2. Sharma M , Ganguly NK : Premature coronary artery disease in Indians and its associated risk factors . Vasc Health Risk Manag 2005 , 1 : 217 - 225 .
3. Kovacic JC , Moreno P , Hachinski V , Nabel EG , Fuster V : Cellular senescence, vascular disease, and aging: part 1 of a 2-part review . Circulation 2011 , 123 : 1650 - 1660 .
4. Lakatta EG , Levy D : Arterial and cardiac aging: major shareholders in cardiovascular disease enterprises: part I: aging arteries: a set up for vascular disease . Circulation 2003 , 107 : 139 - 146 .
5. Hamburg NM , Charbonneau F , Gerhard-Herman M , Ganz P , Creager MA : Comparison of endothelial function in young men and women with a family history of premature coronary artery disease . Am J Cardiol 2004 , 94 : 783 - 785 .
6. Asahara T , Takahashi T , Masuda H , Kalka C , Chen D , Iwaguro H , Inai Y , Silver M , Isner JM : VEGF contributes to postnatal neovascularization by mobilizing bone marrow-derived endothelial progenitor cells . EMBO J 1999 , 18 : 3964 - 3972 .
7. Minamino T , Komuro I : Vascular cell senescence: contribution to atherosclerosis . Circ Res 2007 , 100 : 15 - 26 .
8. Minamino T , Komuro I : Vascular aging: insights from studies on cellular senescence, stem cell aging, and progeroid syndromes . Nat Clin Pract Card 2008 , 5 : 637 - 648 .
9. Minamino T , Miyauchi H , Yoshida T , Ishida Y , Yoshida H , Komuro I : Endothelial cell senescence in human atherosclerosis: role of telomere in endothelial dysfunction . Circulation 2002 , 105 : 1541 - 1544 .
10. Hill JM , Zalos G , Halcox JP , Schenke WH , Waclawiw MA , Quyyumi AA , Finkel T : Circulating endothelial progenitor cells, vascular function, and cardiovascular risk . N Engl J Med 2003 , 348 : 593 - 600 .
11. Choi JH , Kim KL , Huh W , Kim B , Byun J , Suh W , Sung J , Jeon ES , Oh HY , Kim DK : Decreased number and impaired angiogenic function of endothelial progenitor cells in patients with chronic renal failure . Arterioscl Throm Vasc 2004 , 24 : 1246 - 1252 .
12. Ghani U , Shuaib A , Salam A , Nasir A , Shuaib U , Jeerakathil T , Sher F , O'Rourke F , Nasser AM , Schwindt B , Todd K : Endothelial progenitor cells during cerebrovascular disease . Stroke 2005 , 36 : 151 - 153 .
13. Grisar J , Aletaha D , Steiner CW , Kapral T , Steiner S , Seidinger D , Weigel G , Schwarzinger I , Wolozcszuk W , Steiner G , Smolen JS : Depletion of endothelial progenitor cells in the peripheral blood of patients with rheumatoid arthritis . Circulation 2005 , 111 : 204 - 211 .
14. Loomans CJ , de Koning EJ , Staal FJ , Rookmaaker MB , Verseyden C : Endothelial progenitor cell dysfunction: a novel concept in the pathogenesis of vascular complications of type 1 diabetes . Diabetes 2004 , 53 : 195 - 199 .
15. Tepper OM , Galiano RD , Capla JM , Kalka C , Gagne PJ , Jacobowitz GR , Levine JP , Gurtner GC : Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures . Circulation 2002 , 106 : 2781 - 2786 .
16. Ferrari AU , Radaelli A , Centola M : Invited review: aging and the cardiovascular system . J Appl Physiol 2003 , 95 : 2591 - 2597 .
17. Cawthon RM : Telomere measurement by quantitative PCR . Nucleic Acids Res 2002 , 30 : e47 .
18. O'Callaghan N , Dhillon V , Thomas P , Fenech M : A quantitative real-time PCR method for absolute telomere length . Biotechniques 2008 , 44 : 807 - 809 .
19. O'Callaghan NJ , Fenech M : A quantitative PCR method for measuring absolute telomere length . Biol Proced Online 2011 , 13 : 3 .
20. Vasa M , Fichtlscherer S , Aicher A , Adler K , Urbich C , Martin H , Zeiher AM , Dimmeler S : Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease . Circ Res 2001 , 89 : E1 - E7 .
21. Eizawa T , Ikeda U , Murakami Y , Matsui K , Yoshioka T , Takahashi M , Muroi K , Shimada K : Decrease in circulating endothelial progenitor cells in patients with stable coronary artery disease . Heart 2004 , 90 : 685 - 686 .
22. Rauscher FM , Goldschmidt-Clermont PJ , Davis BH , Wang T , Gregg D , Ramaswami P , Pippen AM , Annex BH , Dong C , Taylor DA : Aging, progenitor cell exhaustion, and atherosclerosis . Circulation 2003 , 108 : 457 - 463 .
23. Kushner EJ , Van Guilder GP , Maceneaney OJ , Cech JN , Stauffer BL , DeSouza CA : Aging and endothelial progenitor cell telomere length in healthy men . Clin Chem Lab Med 2009 , 47 : 47 - 50 .
24. Scheubel RJ , Zorn H , Silber RE , Kuss O , Morawietz H , Holtz J , Simm A : Agedependent depression in circulating endothelial progenitor cells in patients undergoing coronary artery bypass grafting . J Am Coll Cardiol 2003 , 42 : 2073 - 2080 .
25. Satoh M , Ishikawa Y , Takahashi Y , Itoh T , Minami Y , Nakamura M : Association between oxidative DNA damage and telomere shortening in circulating endothelial progenitor cells obtained from metabolic syndrome patients with coronary artery disease . Atherosclerosis 2008 , 198 : 347 - 353 .
26. Branch A , Florenza AM , ROvellini A , Torri A , Muzio F , MAcor S , Sommariva D : Lowering effects of four stitins on serum triglycerides . Eur J Pharmacol 1999 , 55 : 499 - 502 .
27. Jialal I , Devaraj S , Singh U , Huet BA : Decreased number and impaired functionality of endothelial progenitor cells in subjects with metabolic syndrome: implication for increased cardiovasculr risk . Atherosclerosis 2010 , 211 : 297 - 302 .
28. Liu L , Wen T , Zheng XY , Yang DG , Zhao SP , Xu DY , Lu GH : Remnant like particles accelerate endothelial progenitor cell senescence of endothelial progenitor cells via oxidative stress . Atherosclerosis 2009 , 202 : 405 - 414 .
29. Paul L : Diet, nutrition and telomere length . J Nutr Biochem 2011 , 22 : 895 - 901 .
30. Kim JH , Ko JH , Lee DC , Lim I , Bang H : Habitual physical exercise has beneficial effects on telomere length in postmenopausal women . Menopause 2012 , 19 : 1109 - 1115 .
31. Laufs U , Werner N , Link A , Endres M , Wassmann S , Jrgens K , Miche E , Bhm M , Nickenig G : Physical training increases endothelial progenitor cells, inhibits neointima formation, and enhances angiogenesis . Circulation 2004 , 109 : 220 - 226 .
32. Vafeiadou K , Weech M , Sharma V , Yaqoob P , Todd S , Williams CM , Jackson KG , Lovegrove JA : A review of the evidence for the effects of total dietary fat, saturated, monounsaturated and n-6 polyunsaturated fatty acids on vascular function, endothelial progenitor cells and microparticles . Brit J Nutr 2012 , 107 : 303 - 324 .
33. Satoh M , Minami Y , Takahashi Y , Tabuchi T , Itoh T , Nakamura M : Effect of intensive lipid-lowering therapy on telomere erosion in endothelial progenitor cells obtained from patients with coronary artery disease . Clin Sci (Lond) 2009 , 116 : 827 - 835 .
34. Webber BJ , Seguin PG , Burnett DG , Clark LL , Otto JL : Prevalence of and risk factors for autopsy-determined atherosclerosis among US service members , 2001 - 2011 . JAMA 2012 , 308 : 2577 - 2583 .
35. Levy D : Combating the epidemic of heart disease . JAMA 2012 , 308 : 2624 - 2625 .
36. Nilsson PM , Lurbe E , Laurent S : The early life origins of vascular ageing and cardiovascular risk: the EVA syndrome . J Hypertens 2008 , 26 : 1049 - 1057 .
37. Gupta R , Misra A , Vikram NK , Kondal D , Gupta SS , Agrawal A , Pandey RM : Younger age of escalation of cardiovascular risk factors in Asian Indian subjects . BMC Cardiovasc Disord 2009 , 9 : 28 .