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Cell Aging in Relation to Stress Arousal and
Cardiovascular Disease Risk Factors
Citation
Epel, Elissa S., Jue Lin, Frank H. Wilhelm, Owen M. Wolkowitz, Richard Cawthon, Nancy E. Adler,
Christyn Dolbier, Wendy B. Mendes, and Elizabeth H. Blackburn. 2006. Cell aging in relation
to stress arousal and cardiovascular disease risk factors. Psychoneuroendocrinology 31(3):
277-287.
Published Version
http://dx.doi.org/10.1016/j.psyneuen.2005.08.011
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http://nrs.harvard.edu/urn-3:HUL.InstRepos:3128692
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Psychoneuroendocrinology(2006)31,277–287
www.elsevier.com/locate/psyneuen
2005 CURT P.RICHTER AWARD WINNER
Cell aging in relation to stress arousal
and cardiovascular disease risk factors
Elissa S. Epela,*, Jue Linb, Frank H. Wilhelmc, Owen M. Wolkowitza,
Richard Cawthone, Nancy E. Adlera, Christyn Dolbierd,
Wendy B. Mendesf, Elizabeth H. Blackburnb
aUCSF Department ofPsychiatry, San Francisco, CA, USA
bUCSF Department ofBiochemistryandBiophysics, San Francisco, CA, USA
cDepartment of Clinical Psychology and Psychotherapy, University of Basel,Basel, Switzerland
dDepartment ofPsychology, East Carolina University, Greenville, NC, USA
eDepartmentof HumanGenetics, University ofUtah, Salt Lake City, UT, USA
fDepartmentof Psychology, Harvard University, Cambridge, MA, USA
Received10August2005
KEYWORDS Summary We previously reported that psychological stress is linked to and
Telomerase; possibly accelerates cellular aging, as reflected by lower PBMC telomerase and
Telomerelength; shortened telomeres. Psychological stress is a major risk factor for cardiovascular
Stress; disease (CVD), with multiple behavioral and physiological mediators. Telomere
Heartratevariability; shortness has been associated with CVD, but the relationship between low
Stresshormones; telomerase activity, a potential precursor to telomere shortening, and CVD risk
Metabolicsyndrome factors has not been examined in humans. Here we examine whether telomere
length and telomerase in leukocytes are associated with physiological signs of
stress arousal and CVD risk factors in 62 healthy women. Low telomerase activity
in leukocytes was associated with exaggerated autonomic reactivity to acute
mental stress and elevated nocturnal epinephrine. Further, low telomerase
activity was associated with the major risk factors for CVD —smoking, poor lipid
profile, high systolic blood pressure, high fasting glucose, greater abdominal
adiposity—as well as to a composite Metabolic Syndrome variable. Telomere
length was related only to elevated stress hormones (catecholamines and
cortisol). Thus, we propose that low leukocyte telomerase constitutes an early
marker of CVD risk, possibly preceding shortened telomeres, that results in part
from chronic stress arousal. Possible cellular mechanisms by which low
telomerase may link stress and traditional risk factors to CVD are discussed.
Abbreviations BMI, body mass index; CVD, cardiovascular disease; HRV, heart rate variability; PBMC, peripheral blood
mononucleocytes;ANCOVA,analysisofcovariance.
*Correspondingauthor.UCSFDepartmentofPsychiatry,3333CaliforniaStreet,Suite465,SanFrancisco,CA,USA.Tel.:C1415476
7648;fax:C14154767744.
E-mailaddress:[email protected](E.S.Epel).
0306-4530/$-seefrontmatterQ2005ElsevierLtd.Allrightsreserved.
doi:10.1016/j.psyneuen.2005.08.011
278 E.S. Epel et al.
These findings may implicate telomerase as a novel and important mediator of
the effects of psychological stress on physical health and disease.
Q 2005 Elsevier Ltd. All rights reserved.
1. Introduction longevity. Shorter telomeres in human leukocytes
have been linked to age-related conditions and
We recently reported that chronic psychological diseases including hypertension, greater pulse
stress was associated with both shorter telomere pressure, hypercholesterolemia (reviewed in Ser-
length and lower telomerase activity in leukocytes rano and Andres, 2004), and increases in insulin
(Epel et al., 2004). Telomeres are DNA-protein resistance (Gardner et al., 2005). Moreover, in the
complexesthatcapchromosomalendsandpromote only prospective study of telomere length and
chromosomal stability. Telomere length is the longevity, telomere length predicted earlier mor-
downstreamoutputoftheactionsofbothlengthen- tality, particularly from infections and CVD (Caw-
ingandshorteningactivitiesinthecell(Blackburn, thon et al., 2003). It is unknown exactly how
2000).Mostnotably,thecellularenzymetelomerase telomere maintenance might influence CVD pro-
adds telomeric repeat sequences to the chromo- cesses. Telomerase increases telomere length.
somalDNAends,preservingnotonlytelomerelength Although telomerase has not been examined in
but also healthy cell function, prolonged stem cell relation to CVD risk in humans, telomerase insuffi-
proliferation, and long-term immune function, ciencyleadstobonemarrowfailure(Marroneetal.,
independently of telomere length (Kim et al., 2005),whileinanimalstudiesandinvitro,telomer-
2003;Lyetal.,2005;Marroneetal.,2005). ase insufficiency has been linked to cardiovascular
Stressarousalislikelytobeonepathwaythrough pathobiology (Yang et al., 1999; Oh, 2003). These
which chronic stress may impact cellular aging findings raise the possibility that telomerase may
(Sapolsky, 2004). Chronic life stress can lead to playanimportantroleinearlydevelopmentofCVD
overexposure to stress responsive adrenal hor- inhumans.
mones, primarily cortisol and catecholamines, or Given the inter-relationships between stress,
tohyporesponsiveness,alsoreflectingdysregulation telomerelengthandCVDriskfactors,inthepresent
(McEwen, 1998). In addition to adrenal hormones, study we also examined the relationship between
autonomic nervous system reactivity to stress, cellular aging and CVD risk factors. We tested
particularly heart rate variability, can serve as a whether telomere length and telomerase were
physiological index of stress vulnerability, and is related to early development of the top CVD risk
related to anxiety (Porges, 1995; Friedman and factors, in a sample of healthy young women (i.e.
Thayer,1998).Wethusexaminedwhethertelomer- those without frank disease). Further, many CVD
ase levels and telomere length in peripheral blood risk factors tend to be correlated, representing
mononucleocytes (PBMCs) are associated with two underlying insulin resistance, labeled as the Meta-
indices of stress arousal—nocturnal excretion of bolic or Insulin Resistance Syndrome (Reaven,
stress hormones (catecholamines and cortisol) and 1988). The Metabolic Syndrome tends to accrue
autonomic nervous system stress reactivity to a with aging and is a well-established precursor to
standardized stressor (vagal tone or heart rate chronic diseases, most notably CVD (Lakka et al.,
variability). 2002). Therefore, we examined whether cell aging
Psychologicalstresshasrecentlyemergedasone waslinkedtothisunderlyingsyndrome,inaddition
of the major risk factors for CVD. The largest toindividualriskfactors.Wespecificallytestedthe
epidemiological study of risk factors for CVD to hypothesis that women with greater levels of
date, with almost 30,000 participants, across 52 cellular aging (i.e. shorter PBMC telomere length
countries, identified the top six modifiable risk and lower telomerase activity) would have greater
factors: cigarette smoking, poor lipid profile, high stress arousal and CVD risk factors, including level
bloodpressure,diabetes,abdominalobesity,andan of MetabolicSyndrome.
indexofpsychosocialstress(Yusefetal.,2004).The
cellular mechanisms by which many of these
2. Materials and methods
physiological and behavioral risk factors, particu-
larly stress, can lead to disease are not well
understood. 2.1. Study overview
However,thereisnowconvergingevidencefrom
basic and clinical studies that telomere mainten- Sixty-twohealthywomen(allmothers),aged20–50,
ance plays an important role in organismal were assessed on mood, life stress, acute mental
Telomerase, stress arousal, and cardiovasculardisease risk 279
stress reactivity in response to a standardized first 7daysof thefollicularphaseofthe menstrual
laboratorystressor,andhealthparameters,includ- cycle,whenestrogenandprogesteronearelowest.
ingablooddraw.Within1weekafterthelaboratory For those taking oral contraceptives, in the
assessment,participantscompletedquestionnaires majority of cases, women participated during the
assessingstress(PerceivedStressScale)(Cohenand ‘placebo week’ when hormone levels are also
Williamson, 1988), tendency to experience nega- lowest.
tivemood(PositiveandNegativeAssessmentScale) Participants fasted for 10h, starting from after
(Watson et al., 1988), years of education com- dinner the previous night, and had a fasting blood
pleted, and health behaviors, and completed an draw around 0800h on the test day. Additional
overnight collection of urine from home, to be assessmentsincludedsmokingstatus,restinghemo-
assayed for stress hormones. dynamics,adiposity,bloodinsulin,glucoseandlipid
profile.PBMCswereisolatedimmediatelyfromthe
blood, and frozen at K808C. Telomerase activity
2.2. Participants and recruitment
levels and telomere length were measured from
frozen cells (O80% viability; telomerase activity
Participants were initially recruited through their
levels measured are shown corrected to arbitrary
child’s health care professional in clinics in Bay
units per viable cell). All participants were then
Area hospitals, or by public postings. To capture a
exposed to a standardized laboratory stressor,
range of stress levels, the sample included both
whileautonomicnervoussystemreactivity(cardiac
mothers of chronically ill children and mothers of
vagal control or high frequency heart rate varia-
healthy children. All women had at least one
bility) was measured repeatedly. Detailed pro-
biological child, and of these participants, 40
ceduresare described below.
were mothers of children with a neurological or
gastrointestinal disorder, or a pervasive develop-
ment disorder (i.e. autism), and 22 had healthy 2.4. Standardized stressor
children (for more detail, see (Epel et al.,
2004).Thisisthesamesampleofstudyparticipants Participantswereexposedtoanadaptedversionof
for which relationships between chronic psycho- the standardized Trier Social Stress Test (Kirsch-
logical stress and telomere length, telomerase and baum et al., 1993). They first rested for 30min
oxidative stress were previously reported(Epel beforesittingbaselinemeasurementsofautonomic
et al., 2004), (with the addition of four additional arousal were taken for 5min. Participants were
participants who had measures of telomerase and giventaskinstructions,andthenexposedto15min
other parameters but not telomere length). Telo- of stressful tasks, including a 5-min speech prep-
meraseactivityandtelomerelengthinPBMCswere aration period, deliverance of a 5-min videotaped
measured as described previously (Epel et al., speech with a live audience of two people, and a
2004). The 62 participants were free of any acute 5-minperformanceofserial subtractionof aprime
orchronichealthconditionsbyselfreport,withthe numberfromahighnumber.Theyperformedthese
exception of controlled hypertension (nZ2) and tasks while seated to limit movement that could
controlled hypothyroidism (nZ1). Participants did affectthe psychophysiology recordings.
nottakeanyotherregularmedications,exceptoral
contraceptives(nZ19,30.6%),andantidepressants
2.4.1.Cardiovascular and autonomic reactivity
(nZ4). All women were currently menstruating.
Cardiovascular beat-to-beat data were assessed at
Nine percent currently smoked (nZ5), but
baseline, during mental stress, including the
refrained from smoking the morning of the blood
anticipation/preparation period, speech stressor,
draw. Participants did not consume more than
and arithmetic stressor. An automatic arm cuff
seven alcoholic beverages per week, or exercise
inflation blood pressure monitor (model SD-700S,
more than 1h a day. They refrained from alcohol
IBS, Waltham, MA) was triggered 4min after onset
the day before the study and during the study day.
ofeachtask,andsystolicanddiastolicvalueswere
recorded on a log sheet. A customized computer
2.3. Laboratory session program written in MATLAB (Wilhelm et al., 1999)
was used for computation of heart rate and high
The laboratory sessions took place at the Oakland frequency HRV (also termed respiratory sinus
Children’s Hospital Pediatric Clinical Research arrhythmia,RSA,anindexofcardiacvagalcontrol).
Center. Efforts to minimize hormonal variability Thebeat-to-beatvaluesofinter-beatintervals(IBI)
from menstrual cycle stages included timing were edited for outliers due to artifacts or ectopic
sessions as follows: Sessions occurred during the myocardial activity, linearly interpolated, and
280 E.S. Epel et al.
converted into instantaneous time series with a intra- and interassay CVs were 10.2 and 15.0%,
resolution of 4Hz. IBI series were linearly respectively. Cortisol was measured by a radio-
detrended, and the power spectral densities immunoassay. The sensitivity of the method is
derived for each experimental period using the 10nml. Intra- and interassay CVs were 9.3 and
Welchalgorithm,whichensembleaveragessucces- 5.4%, respectively. Creatinine was measured
siveperiodograms(atotalof9240-pointsegments, spectrophotometrically by the Jaffe reaction at
overlapping by 50%, were Hanning windowed, zero 490nm after extraction of the samples with ethyl
paddedtolength256,andsubjectedtoFastFourier ether to remove interfering substances.
transform; estimates of power were adjusted to
account for attenuation produced by the Hanning
2.6. Cardiovascular disease risk
window). High-frequency HRV was computed by
and metabolic syndrome composite
summation of power spectral density values in the
0.15–0.50Hz frequency range, and resulting values
Weight was measured using a balance beam scale,
were normalized using the naturallogarithm.
and height with a stadiometer. BMI was calculated
as weight (kg)/height (m)2. Saggital diameter was
2.5. Urine sampling measured with a meter stick with a leveler on the
top, as the widest horizontal diameter from the
A subsample of 41 participants collected a 12h lowerlumbartothebelly,ameasurewhichishighly
nocturnal urine sample which was assayed for correlated with visceral fat from CT scans (Kvist
stress hormones (cortisol, epinephrine, and nor- et al.,1988).
epinephrine) and creatinine on one night in the Lipid levels were analyzed in triplicate in the
week following the session, typically from 2000 CoreLaboratoryoftheGCRCattheMedicalCollege
to 0800h (to assess resting arousal, minimizing ofWisconsin(RR00058).Fastingplasmatriglyceride
contributions from daytime activity levels). and cholesterol levels were determined spectro-
Selection of the subsample was based simply on photometricallyusingkitsfromStanbioLaboratory,
the timing of implementation of the urine Inc. (San Antonio, TX), and Roche Diagnostics
collection (the participants run in the first few (Indianapolis, IN), respectively. HDL-cholesterol
months did not collect urine). The urine was kept was determined after phosphotungstic acid/MgCl2
in a cooler with ample ice blocks for the duration precipitation(SigmaDiagnostics,StLouis,MO).The
of the collection, and picked up promptly the respective intraassay and interassaycoefficients of
next morning. Aliquots were taken and frozen at variationforthelipidanalyseswere4.7and5.3%for
K808C until batch analysis (to minimize inter- triglyceride, 5.5 and 6.7% for cholesterol, and 5.7
assay variation). The range of collection times and6.1%forHDL-cholesterol.LDLcholesterollevel
was from 11.75 to 12.75h, with 90% of the was derived with the formula Total Cholesterol—
sample collecting for a full 12h. Urinary (0.20*TriglycerideCHDL). Insulin and glucose were
hormones were analyzed as ng/mg creatinine, measured in the Cornell GCRC, using standard
to adjust for differences in durations of collec- methods with commercial kits.
tions and in body mass. Participants who had The ‘National Cholesterol Education Program
collection volumes less than 300ml (nZ4) were (NCEP) Adult Treatment Panel III (ATPIII)’ defi-
determined to have incomplete collections and nition for Metabolic Syndrome includes scoring in
their urinary hormone values were excluded from the elevated range on at least three of the
analyses, as done in other studies with 12h urine following symptoms: abdominal obesity, triglycer-
collections (e.g. Greendale et al., 1999). Values ides, low HDL, high blood pressure, and high
were normally distributed, and results were fasting glucose (NCEP, 1999). To examine the
similar when using a natural log transformation subclinical level of cumulative risk factors in a
(raw values are shown). Assays were done in the healthy sample who would not meet threshold for
laboratory of Dr Robert Chatterton, Northwestern full blown Metabolic Syndrome, we examined the
University. Urinary epinephrine and norepi- level of Metabolic Syndrome by creating a
nephrine were assayed with kits provided by continuous measure of how high one is on each
American Laboratory Products Company (Alpco), of these five factors. Each factor, waist circum-
Windham, NH. The sensitivity is 0.33 and 1.33ng/ ference, HDL cholesterol, triglycerides, systolic
ml, respectively, for epinephrine and norepi- blood pressure, and glucose levels, was standar-
nephrine. For epinephrine the intra- and inter- dized, and a mean was taken. Higher mean z
assay coefficients of variation (CV’s) were 7.2 and scores represent a greater extent of Metabolic
15.4%, respectively. For norepinephrine, the Syndrome.
Telomerase, stress arousal, and cardiovasculardisease risk 281
3. Statistical analyses
Our primary goal was to test whether women with
differing levels of cellular aging differed in stress
arousal and CVD risk factors. Participants were
categorized into high and low telomerase activity
groups, and high and low telomere length groups,
based on mean splits. T-tests, with 1-tailed
significance tests for a priori hypotheses, were
performed to test for any differences between
these cell aging groups. To follow up these
analyses, we tested whether findings were inde-
pendentofthetwomajorconfoundingfactors,age
and BMI, by performing analyses of covariance
(ANCOVAs) adjusting for these factors with these
covariates.Therewerenosignificantdifferencesin
telomerase activity or telomere length overall
between the caregiving vs. control groups, and
thusthesewomenwereexaminedasonegroup.All
variables were examined for normal distributions,
andtransformedwhenappropriate(i.e.telomerase
was slightly positively skewed, and thus log
transformed).
Figure 1 Nocturnal urinary stress hormones (ng/ml
creatinine, standardized values), adjusted for BMI and
4. Results age. (A) Low and high telomerase activity groups. (B)
Shortandlongtelomerelengthgroups.*Zp!.05,levels
arehighercomparedtolevelsofthesamehormoneinthe
4.1. Stress arousal
other(high/long)group.
Wefoundthatlowertelomeraselevelswererelated
toneuroendocrineandpsychosocialdataindicative
compared to the high telomerase group (Fig. 2).
of greater life stress. ANCOVAs showed that the
Average HRV during stress controlling for baseline
womenwithlowtelomerasehadsignificantlyhigher
HRV,age,andBMIwaslowerinthelowtelomerase
excretion of nocturnal epinephrine (p!.01), and group: F(1,40)Z3.85,p!.028).
marginally higher norepinephrine (p!.08) but
Telomere length, unlike telomerase, was not
similarlevelsofcortisol(SeeFig.1forstandardized
related to negative mood, education, or HRV.
values, adjusted for BMI and age). The low
Shortertelomerelengthwas,however,significantly
telomerase group was not significantly different in
relatedtohigherlevelsofallthreestresshormones
negative affect, although when examined as a
measured (cortisol, epinephrine, and norepi-
continuous variable, telomerase (log transformed)
nephrine), after controlling for BMI and age (p’s!
was correlated with trait negative mood (usingthe
.05, see Fig. 1for standardized levels).
PANAS) (rZK.31, p!.02), and as reported else-
where, with perceived stress and chronicity of
stress (Epel et al., 2004). Low telomerase was also 4.2. Cardiovascular disease risk
associatedwithfeweryearsofeducation(Table1),
whichisitselfariskfactorforCVD,inpartthrough AsshowninTable1,t-testsrevealthatalthoughage
conferring greater exposure to chronic stress was similar between the high and low telomerase
(Wamala etal., 1999). groups, the low telomerase group had significantly
Toexamineautonomicreactivitytostress(heart greater BMI and abdominal adiposity (saggital
rate, blood pressure, and HRV), repeated measure diameter), and higher resting heart rate, systolic
ANCOVAs were performed, controlling for baseline blood pressure, fasting glucose, LDL and total
values of each dependent variable, BMI, and age. cholesterol, total/HDL cholesterol ratio, and a
Groups were similar in blood pressure and heart greater tendency toward lower resting HRV. They
ratereactivity.However,thelowtelomerasegroup werealsomorelikelytobesmokers;althoughthere
respondedtostresswithgreaterdecreasesinHRV, wereonlyfivecurrentsmokers,allwereinthelow
282 E.S. Epel et al.
Table1 Rawmeansandstandarderrorsofage,education,andCVDriskfactorsinhighandlowtelomerasegroups.
Hightelomerase Lowtelomerase t-testvalues,sig- Significance
M(SE)NZ29 M(SE)NZ33 nificance(1- afteradjust-
tailed) ingforBMI,
age(ANCOVA)
Age(years) 39.4(1.0) 37.2(1.2) K1.41,p!.14 N/A
Education(years) 15.50(0.35) 14.5(0.33) K2.02,p!.025 N/A
Adiposity
Bodymassindex(BMI;kg/m2) 23.99(0.68) 27.44(1.12) 2.55,p!.007 N/A
Sagittaldiameter(cm) 19.22(0.88) 22.09(1.01) 2.12,p!.019 N/A
Restingcardiovascularactivitya
Systolicbloodpressure(mmHg) 105.04(1.79) 109.83(1.60) 2.00,p!.025 p!.150
Diastolicbloodpressure(mmHg) 64.96(1.85) 68.10(1.53) 1.36,p!.09 p!.330
Heartrate(bpm) 76.56(1.90) 82.58(1.85) 2.27,p!.015 p!.041
Highfrequencyheartratevaria- 6.40(0.11) 6.15(0.09) K1.67,p!.051 p!.054
bility(ms2)
Cholesterollevels
HDL(mg/dl) 50.11(1.96) 45.93(2.55) K1.30,p!.095 p!.300
LDL(mg/dl) 119.11(6.39) 142.97(9.46) 2.37,p!.011 p!.012
Total(mg/dl) 184.14(7.0) 203.31(8.56) 1.72,p!.045 p!.016
Total/HDLratio 3.77(0.17) 4.83(0.34) 2.73,p!.005 p!.017
Fastinginsulin(uIU/mL) 12.33(1.56) 11.81(1.29) K0.26,p!.395 p!.463
Fastingglucose(mg/dL) 84.33(0.94) 88.69(1.40) 2.55,p!.007 p!.008
Metabolicsyndrome K.21(.09) .13(.09) 2.65,p!.006 p!.09
(meanz-score)
N/AZnotapplicable.
a Cardiovascularresultsareafterexcludingthetwosubjectsonantihypertensivemedications.
telomerase group (Chi square of telomerase group CVD in large samples (e.g. Yusef et al., 2004),
by smoking group: X2(1)Z4.77, p!.015), falling but the intermediate mediators of these relation-
within the bottom 15% of all telomerase values ships have not been characterized. The present
(Fig. 3). study represents a first test of whether leukocyte
Because obesity both may be linked to low aging might potentially underlie the relationship
telomerase (via unknown pathways) and also could between stress and CVD risk factors. We found
generate the other risk factors, BMI (and age) that telomere shortness and low telomerase are
were controlled for using ANCOVAs. As shown in both associated with stress arousal (nocturnal
Table 1, differences between the low and high stress hormones), and low telomerase is associ-
telomerase groups in systolic blood pressure and ated with several risk factors for CVD, as
Metabolic Syndrome composite became non-sig- discussed below. It is likely that stress hormones
nificant and thus were partially mediated via BMI play an important role in the pathway to cellular
(age was not different between groups, only BMI). aging. These findings support a tentative role for
In contrast, heart rate, lipids, and fasting glucose telomerase in the pathway between stress and
differences remained significant and hence were
disease. Further research is needed to determine
independent of BMI. We performed the same
the mechanisms of how the telomerase/telomere
analyses with telomere length groups but did not
maintenance system might play a causal role in
observe associations between telomere length and
CVD, whether stress is an early precursor, and
CVD risk.
how genetic predispositions might promote vul-
nerability to these related factors (stress arousal,
cellular aging).
5. Discussion
5.1. Stress arousal
Chronic psychological stress has been associated
both with signs of accelerated leukocyte aging in This is the first clinical study to our knowledge to
our current sample (Epel et al., 2004) and with document relationships between stress hormones
Telomerase, stress arousal, and cardiovasculardisease risk 283
Figure 2 High frequency HRV by telomerase groups.
Autonomic nervous system function at baseline and
duringstressfultasksforlowandhightelomerasegroups. Figure 3 Telomerase levels among smokers and non-
Asshownabove,acrossalltimepoints,aquadraticmodel smokers.Thefivesmokershadsignificantlyloweraverage
bestfitsthedata,F(1,41)Z7.81,p!.004,showinglower telomerasethanthenon-smokers,t-testZ3.26,p!.002.
baselinebutalsoadecreaseinHRVduringthestressorin
the low telomerase group only. Two subjects with
controlled hypertension were excluded (all values
adjusted for age and BMI). Base, baseline; Prep, (negativemood,lowereducation,autonomicarou-
preparation/anticipation of speech; Speech, speech sal),aswellasCVDriskfactors,andalackofsimilar
task;Math,mathtask;Recovery,restperiod. associations for telomere length. In terms of
autonomic arousal, women with lower telomerase
activity levels tended to have higher resting heart
with any markers of cellular aging in people (or rate and somewhat lower resting cardiac vagal
in vitro). Telomerase status and telomere length controlandgreatervagalsuppression(decreasesin
are well established indices of cellular health and high frequency HRV) in response to a standardized
longevity(Blackburn,2000).Hereweshowthatlow laboratorystressor,allfindingsindependentofage
telomeraseandshorttelomeresarebothrelatedto and BMI. Chronically suppressed vagal control may
elevations in nocturnal excretion of epinephrine indicate impaired allostasis and restorative pro-
(and short telomeres to elevations in norepi- cesses, and increased risk for CVD, whereas vagal
nephrineandcortisol).Theseresultsareconsistent suppression in response to acute stressors is a
withourearlierfindingsthatgreaterperceptionand physiological indicator of psychological stress
duration of psychological stress are related to vulnerability, at least in infants (Porges, 1995;
shorter telomere length and decreased telomerase Carney et al., 2001). Since parasympathetic
activity (Epel et al., 2004). nervous system activity controls restorative pro-
While both low telomerase and short bulk cesses,itmaybeparticularlyimportantinpromot-
telomere length were related to higher stress ing longevity.
hormones, mainly epinephrine, the remaining Thedirectionofcausalityforthepresentfindings
tests showed significant associations for telomer- is unknown. Stress and associated stress arousal
ase, but not for telomere length. There is a may contribute to decreased telomerase activity,
consistent pattern linking telomerase activity to sincetheoppositedirectionofcausalityseemsless
additional indices of stress and stress arousal probable. In vitro, catecholamines can reduce
284 E.S. Epel et al.
oxidative stress in certain neurons (Troadec et al., to reduced cell growth (Minamino and Kouremba-
2001) but, more relevant to this study, peripheral nas, 2001). Telomerase regulates mitosis and
infusions of catecholamines increase oxidative longevity of endothelial and progenitor endo-
stress inrats(Aizawa et al., 2002). thelial cells (Yang et al., 1999; Murasawa et al.,
Glucocorticoidsarelinkedtoneuronalcelldeath 2002). Telomerase could also play a significant
(Sapolsky, 1996), in part through greater oxidative role in early disease processes independently of
stress damage, by increasing intra-neuronal levels telomere length. Telomerase allows cells to grow
ofglutamateandcalciumandimpairingantioxidant healthily, even those with short average telomere
defenses (McIntosh and Sapolsky, 1996). Glucocor- length (Kim et al., 2003). Telomerase is protec-
ticoid administration increases plasma markers of tive of telomeric DNA even for long telomeres,
oxidative stress in mammals (Orzechowski et al., preventing them from occasional catastrophic
2002;Linetal.,2004).Itisunclearhowthesestress shortening that can cause cell growth arrest or
hormonesmightaffecttelomeraseandtelomeresin genomic instability (Chan and Blackburn, 2003).
human PBMCs. The relationships between stress Telomerase promotes stem cell proliferation even
hormones and telomerase activity in vitro are in the absence of any telomerase enzymatic
currently under investigation (J.L. and E.H.B., functional capability (Sarin et al., in press).
unpublished results).
5.2.2. Telomerase reflecting CVD processes
Telomerase could also be a marker, rather than
5.2. Telomerase and CVD risk
causal factor, of CVD risk. Low PBMC telomerase
activity could reflect overall organismal low telo-
Low telomerase activity was consistently associ-
merase activity, including low telomerase in
ated with proxy markers for the top six major risk
cardiovascular tissue. Thus, low telomerase may
factors for CVD (Yusef et al., 2004): smoking,
simply be an early cellular indicator of disease
abdominal adiposity, lipids, fasting glucose, blood
processes.Earlyfactors,suchasprenatalstressand
pressure, and psychosocial stress. Telomerase was
nutrition, can pre-program aspects of physiology
alsoassociatedwithaMetabolicSyndromecluster,
(obesity, insulin resistance, hypothalamic pituitary
although this relationship was diminished after
adrenal responsiveness), causing later Metabolic
controlling for BMI, and became of borderline
Syndrome (McMillen and Robinson, 2005). Such
significance.
early developmental factors might also affect
Low telomerase, through an unidentified path-
trajectories of cellular aging as well, by limiting
way, could plausibly lead to increases in the
the total replenishment capabilities of cells. Such
Metabolic Syndrome, the Metabolic Syndrome
limitation is seen in patients haploinsufficient for
factors could lead to decreased telomerase, or,
telomerase, who show premature bone marrow
most likely, bidirectional pathways exist, as
stem cell exhaustion (Vulliamy et al., 2001;
described below. Finally, both may be indepen-
Marrone et al., 2005).
dently related to athird factor.
5.2.3. CVD processesdecreasing telomerase
5.2.1. Lower telomerasecontributing The Metabolic Syndrome, through obesity or
toCVD processes insulin resistance, may accelerate cell aging
Low telomerase and its predicted eventual out- processes by causing, for example, greater
come, shortened bulk telomeres, potentially exposure to hyperglycemia (Blazer et al., 2002),
prevent the long-term tissue replenishment insulin (Gardner et al., 2005) and oxidative stress
necessary for endothelial and smooth muscle associated with insulin resistance (Davi et al.,
cardiovascular cells, contributing to CVD risk. 2005). Given the relations we report here
Low telomerase in leukocytes may reflect low between one-time measurement of leukocyte
telomerase in cardiovascular cells as well, telomerase activity level and stable factors such
although this has not been explored. Prior as one’s body mass and cholesterol, it appears as
evidence, although limited to animal or in vitro if snap–shot telomerase levels reflect relatively
studies, suggests that low telomerase is associ- stable levels over time. This may be especially
ated with cardiovascular cell viability. In cardio- true in the current study given that no partici-
myocytes, overexpression of the protein pants were ill, and that the collection conditions
component of telomerase (hTERT) inhibits apop- were highly standardized, in terms of time of
tosis in culture (Oh, 2003). In vascular smooth day, fasting state, and stage of menstrual cycle.
muscle cells, telomerase is related to cell Prolonged relative elevations of blood glucose
division, whereas telomerase inhibition is related levels, even within the normal non-diabetic range
Telomerase, stress arousal, and cardiovasculardisease risk 285
(range of 71–107 in this sample), might down- they were not related among those under 30
regulate telomerase activity or expression, and years old (rZK.15, ns) (nZ9). If it is true that
hence promote cell senescence, regardless of telomerase is related to shorter telomeres mainly
telomere length (Chan and Blackburn, 2003; Kim with advancing age, then over time, we hypoth-
et al., 2003). Thus low telomerase may be part esize that insufficient telomerase will lead to
of the cellular events that promote both early shorter telomeres, reduced replenishment of
disease as well as explain how later these risk tissues, decreased efficiency of endothelial and
factors lead to frank CVD. Prospective cohort vascular functioning, and frank disease.
studies are needed to indicate whether low
leukocyte telomerase activity should be included
among markers for CVD risk.
6. Summary
5.2.4.TelomerelengthandCVDrisk:nullfindings
We found no relationship between mean bulk
Our results show, for the first time, that lower
telomere length (long vs. short telomere groups,
levels of PBMC telomerase activity are associated
or by using correlations) and CVD risk. This might
with increased excretion of stress hormones and
be because of the relatively young healthy
well-established physiological and behavioral risk
sample analyzed in our study, or because
factors for CVD in a healthy sample. While in vitro
telomere length is associated with CVD through
studies have suggested a crucial function of
non-traditional pathways (i.e. independent of
telomerase in the health of cardiovascular-related
common CVD risk factors). In one study, short
cells, this is the first human study to show
telomere length conferred a three-fold risk of
links between telomerase and major CVD risk
premature myocardial infarction, but this
factors. Interestingly, telomere length was not
relationship was independent of established risk
associated with CVD risk factors in our relatively
factors (blood pressure, smoking, lipids, and
young cohort. Telomere length is driven upward
inflammation) (Brouilette et al., 2003). Lastly,
by telomerase activity. Our finding that low
the failure to discern a relationship could be due
telomerase is associated with the major risk
to methodological limitations for telomere length
factors for CVD, prior to any onset of clinically
measurement. The critical shortening of even one
manifested CVD or telomere shortening, suggest
telomere may trigger premature senescence of a
that the telomere shortness previously reported in
cell. Hence, average bulk telomere length in a
CVD (Serrano and Andres, 2004) may be a direct
relatively young and healthy sample may not be
result of previously lower telomerase activity.
sensitive enough to detect early changes, such as
Thus, in younger individuals, such as the 20–50
single strand DNA breaks in the telomere or rare
year-old cohort we studied, telomerase activity
catastrophic shortening events that occur when
may be an earlier marker of preclinical disease
telomerase is deficient (Chan and Blackburn,
risk than telomere length, possibly acting as a
2003), which would affect a cell’s longevity. In
harbinger of subsequent telomere shortening that
addition, most risk factors in this sample, such as
manifests later in life.
blood pressure and fasting glucose, were in the
These findings offer an important clue into
subclinical range, as might be expected given the
young age and health of the sample. Epidemio- mechanisms—both of how telomere shortening is
logical studies are needed to determine whether linked to stress and CVD, and possibly of how
our null findings are due to the lack of clinical traditional risk factors promote CVD. It may be
morbidity or relatively young age of the current highly clinically significant that obesity and
sample. Since telomere length was not correlated smoking are linked to decreased telomerase.
with CVD risk factors over the whole sample, These common risk factors could impair telomer-
telomerase may be a separate indicator from ase enzyme function or interfere with its
telomere length, or more likely, an earlier expression. Perhaps most significantly, our find-
indicator than telomere length; of cell aging. ings suggest early precursors of low telomerase
Eventually, with increasing age, if telomerase activity—neuroendocrine and autonomic stress
activity indeed reflects stable levels, telomere arousal. These findings also open up the possi-
length may become shortened in the low bility that telomerase may be a useful marker for
telomerase activity group. Supporting this monitoring health, such as a barometer for
hypothesis, telomerase and telomere length examining effects of lifestyle and psychosocial
were marginally related (rZC.23, p!.06) in interventions on cardiovascular health. There is
the 30–50 year old participants (nZ47), but yet much to be learned about human health from